Feline Fc epsilon receptor alpha chain proteins and therapeutic uses thereof

ABSTRACT

The present invention relates to feline Fc epsilon receptor alpha chain nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes methods to detect IgE using such proteins and antibodies. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to mediate Fc epsilon receptor-mediated biological responses.

FIELD OF THE INVENTION

The present invention relates to feline Fc epsilon receptor alpha chainnucleic acid molecules, proteins encoded by such nucleic acid molecules,antibodies raised against such proteins, and inhibitors of suchproteins. The present invention also includes methods to detect IgEusing such proteins and antibodies.

BACKGROUND OF THE INVENTION

Diagnosis of disease and determination of treatment efficacy areimportant tools in medicine. IgE antibody production in an animal can beindicative of disease including, for example, allergy, atopic disease,hyper IgE syndrome, internal parasite infections and B cell neoplasia.In addition, detection of IgE production in an animal following atreatment is indicative of the efficacy of the treatment, such as whenusing treatments intended to disrupt IgE production.

Immunological stimulation can be mediated by IgE antibodies when IgEcomplexes with Fc epsilon receptors. Fc epsilon receptors are found onthe surface of certain cell types, such as mast cells. Mast cells storebiological mediators including histamine, prostaglandins and proteases.Release of these biological mediators is triggered when IgE antibodiescomplex with Fc epsilon receptors on the surface of a cell. Clinicalsymptoms result from the release of the biological mediators into thetissue of an animal.

Until the discovery of the present invention, detection of IgE insamples obtained from animals has been hindered by the absence ofsuitable reagents for detection of IgE. Various compounds have been usedto detect IgE in IgE-containing compositions. In particular, antibodiesthat bind selectively to epsilon idiotype antibodies (i.e., anti-IgEantibodies) have been used to detect IgE. These anti-IgE antibodies,however, can cross-react with other antibody idiotypes, such as gammaisotype antibodies. Also, creation of reagents capable of inhibiting theactivity of Fc epsilon receptors has been limited.

The discovery of the present invention includes a novel feline Fcepsilon receptor alpha chain (FcεRα) protein and the use of such aprotein to detect the presence of IgE in a putative IgE-containingcomposition; to identify inhibitors of biological responses mediated bya feline FcεRα protein; and as a therapeutic compound to prevent ortreat clinical symptoms that result from feline FcεRα-mediatedbiological responses. When used in an assay to detect IgE, a felineFcεRα protein provides an advantage over, for example anti-IgEantibodies, to detect IgE because a feline FcεRα protein can bind to anIgE with more specificity (i.e., less idiotype cross-reactivity) andmore sensitivity (i.e., affinity) than anti-IgE binding antibodies.

Prior investigators have disclosed the nucleic acid sequence for: thehuman FcεR alpha chain (Kochan et al., Nucleic Acids Res. 16:3584, 1988;Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911, 1988; and Panget al., J. Immunol. 151:6166-6174, 1993); the human FcεR beta chain(Kuster et al., J. Biol. Chem. 267:12782-12787, 1992); the human FcεRgamma chain (Kuster et al., J. Biol. Chem. 265:6448-6452, 1990); and thecanine FcεR alpha chain (GenBank™ accession number D16413). Although thesubunits of human FcεR have been known as early as 1988, they have neverbeen used to identify a feline FcεR. Similarly, even though the canineFcεR chain has been known since 1993, it has never been used to identifya feline FcεR. Moreover, the determination of human and canine Fcepsilon receptor sequences does not indicate, suggest or predict thecloning of a novel FcεRα gene from a different species, in particular,from a feline species.

Thus, products and processes of the present invention are needed in theart that will provide specific detection of IgE and treatment of Fcepsilon receptor-mediated disease.

SUMMARY OF THE INVENTION

The present invention relates to a novel product and process fordetecting IgE and protecting animals from Fc epsilon receptor-mediatedbiological responses. According to the present invention there areprovided feline FcεRα proteins and mimetopes thereof; feline FcεRαnucleic acid molecules, including those that encode such proteins;antibodies raised against such feline FcεRα proteins (i.e., anti-felineFcεRα antibodies); and other compounds that inhibit the ability offeline FcεRα protein to form a complex with IgE (i.e, inhibitorycompounds or inhibitors).

The present invention also includes methods to obtain such proteins,mimetopes, nucleic acid molecules, antibodies and inhibitory compounds.Also included in the present invention are therapeutic compositionscomprising such proteins, mimetopes, nucleic acid molecules, antibodies,and/or inhibitory compounds, as well as use of such therapeuticcompositions to Fc epsilon receptor-mediated biological responses.

One embodiment of the present invention is an isolated nucleic acidmolecule encoding a feline FcεRα protein. The feline FcεRα proteinpreferably includes: proteins comprising amino acid sequences SEQ IDNO:2, SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:13; and proteins encodedby allelic variants of a nucleic acid molecules encoding a proteincomprising any of the amino acid sequences. Particularly preferredfeline FcεRα nucleic acid molecules include: nucleic acid moleculescomprising nucleic acid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ IDNO:15 and SEQ ID NO:16; and nucleic acid molecules comprising allelicvariants of nucleic acid molecules comprising nucleic acid sequences SEQID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.

The present invention also includes an isolated feline FcεRα protein. Apreferrred feline FcεRα protein is encoded by a nucleic acid moleculethat hybridizes under stringent hybridization conditions to a nucleicacid sequence including SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:15 and SEQ ID NO:16. Particularly preferred feline FcεRα proteinsinclude at least one of the following amino acid sequences: SEQ ID NO:2,SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:13.

The present invention also relates to recombinant molecules, recombinantviruses and recombinant cells that include feline FcεRα nucleic acidmolecules of the present invention. Also included are methods to producesuch nucleic acid molecules, recombinant molecules, recombinant virusesand recombinant cells.

The present invention also includes detection methods and kits thatdetect IgE. One embodiment of the present invention is a method todetect IgE comprising: (a) contacting an isolated feline FcεRα moleculewith a putative IgE-containing composition under conditions suitable forformation of a FcεRα molecule:IgE complex; and (b) determining thepresence of IgE by detecting the FcεRα molecule:IgE complex, thepresence of the FcεRα molecule:IgE complex indicating the presence ofIgE. A preferred feline FcεRα molecule is one which a carbohydrate groupof the feline FcεRα molecule is conjugated to biotin.

Another embodiment of the present invention is a method to detect IgEcomprising: (a) contacting a recombinant cell with a putativeIgE-containing composition under conditions suitable for formation of arecombinant cell:IgE complex, in which the recombinant cell comprises afeline FcεRα molecule; and (b) determining the presence of IgE bydetecting the recombinant cell:IgE complex, the presence of therecombinant cell:IgE complex indicating the presence of IgE. A preferredmethod to detect IgE comprises: (a) immobilizing the FcεRα molecule on asubstrate; (b) contacting the FcεRα molecule with the putativeIgE-containing composition under conditions suitable for formation of aFcεRα molecule:IgE complex bound to the substrate; (c) removingnon-bound material from the substrate under conditions that retain FcεRαmolecule:IgE complex binding to the substrate; and (d) detecting thepresence of the FcεRα molecule:IgE complex. Another preferred method todetect IgE comprises: (a) immobilizing a specific antigen on asubstrate; (b) contacting the antigen with the putative IgE-containingcomposition under conditions suitable for formation of an antigen:IgEcomplex bound to the substrate; (c) removing non-bound material from thesubstrate under conditions that retain antigen:IgE complex binding tosaid substrate; and (d) detecting the presence of the antigen:IgEcomplex by contacting the antigen:IgE complex with said FcεRα molecule.Another preferred method to detect IgE comprises: (a) immobilizing anantibody that binds selectively to IgE on a substrate; (b) contactingthe antibody with the putative IgE-containing composition underconditions suitable for formation of an antibody:IgE complex bound tothe substrate; (c) removing non-bound material from the substrate underconditions that retain antibody:IgE complex binding to the substrate;and (d) detecting the presence of the antibody:IgE complex by contactingthe antibody:IgE complex with said FcεRα molecule. Another preferredmethod to detect IgE comprises: (a) immobilizing a putativeIgE-containing composition on a substrate; (b) contacting thecomposition with the FcεRα molecule under conditions suitable forformation of a FcεRα molecule:IgE complex bound to the substrate; (c)removing non-bound material from the substrate under conditions thatretain FcεRα molecule:IgE complex binding to the substrate; and (d)detecting the presence of the FcεRα molecule:IgE complex.

Another embodiment of the present invention is a method to detect fleaallergy dermatitis comprising: (a) immobilizing a flea allergen on asubstrate; (b) contacting the flea allergen with a putativeIgE-containing composition under conditions suitable for formation of anallergen:IgE complex bound to the substrate; (c) removing non-boundmaterial from the substrate under conditions that retain allergen:IgEcomplex binding to the substrate; and (d) detecting the presence of theallergen:IgE complex by contacting said allergen:IgE complex with afeline FcεRα protein. Preferably, the flea allergen is a flea salivaantigen and more preferably flea saliva products and/or flea salivaproteins.

The present invention also includes a kit for performing methods of thepresent invention. One embodiment is a kit for detecting IgE comprisinga feline FcεRα protein and a means for detecting IgE. Another embodimentis a kit for detecting flea allergy dermatitis comprising a feline FcεRαprotein and a flea allergen.

The present invention also includes an inhibitor that interferes withformation of a complex between feline FcεRα protein and IgE, in whichthe inhibitor is identified by its ability to interfere with the complexformation. A particularly preferred inhibitor includes a substrateanalog of a feline FcεRα protein, a mimetope of a feline FcεRα proteinand a soluble portion of a feline FcεRα protein. Also included is amethod to identify a compound that interferes with formation of acomplex between feline FcεRα protein and IgE, the method comprising: (a)contacting an isolated feline FcεRα protein with a putative inhibitorycompound under conditions in which, in the absence of the compound, thefeline FcεRα protein forms a complex with IgE; and (b) determining ifthe putative inhibitory compound inhibits the complex formation. A testkit is also includes to identify a compound capable of interfering withformation of a complex between a feline FcεRα protein and IgE, the testkit comprising an isolated feline FcεRα protein that can complex withIgE and a means for determining the extent of interference of thecomplex formation in the presence of a putative inhibitory compound.

Yet another embodiment of the present invention is a therapeuticcomposition that is capable of reducing Fc epsilon receptor-mediatedbiological responses. Such a therapeutic composition includes one ormore of the following therapeutic compounds: an isolated feline FcεRαprotein; a mimetope of a feline FcεRα protein; an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with afeline FcεRα gene; an isolated antibody that selectively binds to afeline FcεRα protein; and an inhibitor that interferes with formation ofa complex between a feline FcεRα protein and IgE. A method of thepresent invention includes the step of administering to an animal atherapeutic composition of the present invention.

Yet another embodiment of the present invention is a method to produce afeline FcεRα protein, the method comprising culturing a cell transformedwith a nucleic acid molecule encoding a feline FcεRα protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated feline Fc epsilon receptoralpha chain (FcεRα) proteins, isolated feline FcεRα nucleic acidmolecules, antibodies directed against feline FcεRα proteins and otherinhibitors of feline FcεRα activity. As used herein, the terms isolatedfeline FcεRα proteins and isolated feline FcεRα nucleic acid moleculesrefers to feline FcεRα proteins and feline FcεRα nucleic acid moleculesderived from cats and, as such, can be obtained from their naturalsource or can be produced using, for example, recombinant nucleic acidtechnology or chemical synthesis. Also included in the present inventionis the use of these proteins and antibodies in a method to detectepsilon immunoglobulin (referred to herein as IgE or IgE antibody) aswell as in other applications, such as those disclosed below. Theproducts and processes of the present invention are advantageous becausethey enable the detection of IgE and the inhibition of IgE or felineFcεRα protein activity associated with disease. As used herein, felineFc epsilon alpha chain receptor protein can be referred to as FcεRαprotein or FcεRα chain protein.

One embodiment of the present invention is an isolated proteincomprising a feline FcεRα protein. It is to be noted that the term "a"or "an" entity refers to one or more of that entity; for example, aprotein refers to one or more proteins or at least one protein. As such,the terms "a" (or "an"), "one or more" and "at least one" can be usedinterchangeably herein. It is also to be noted that the terms"comprising", "including", and "having" can be used interchangeably.Furthermore, a compound "selected from the group consisting of" refersto one or more of the compounds in the list that follows, includingmixtures (i.e., combinations) of two or more of the compounds. Accordingto the present invention, an isolated, or biologically pure, protein, isa protein that has been removed from its natural milieu. As such,"isolated" and "biologically pure" do not necessarily reflect the extentto which the protein has been purified. An isolated protein of thepresent invention can be obtained from its natural source, can beproduced using recombinant DNA technology or can be produced by chemicalsynthesis.

As used herein, an isolated feline FcεRα protein can be a full-lengthprotein or any homolog of such a protein. As used herein, a protein canbe a polypeptide or a peptide. Preferably, a feline FcεRα proteincomprises at least a portion of a feline FcεRα protein that binds toIgE, i.e., that is capable of forming a complex with an IgE.

A feline FcεRα protein of the present invention, including a homolog,can be identified in a straight-forward manner by the protein's abilityto bind to IgE. Examples of feline FcεRα protein homologs include felineFcεRα proteins in which amino acids have been deleted (e.g., a truncatedversion of the protein, such as a peptide), inserted, inverted,substituted and/or derivatized (e.g., by glycosylation, phosphorylation,acetylation, myristoylation, prenylation, palmitoylation, amidationand/or addition of glycerophosphatidyl inositol) such that the homologis capable of binding to IgE.

Feline FcεRα protein homologs can be the result of natural allelicvariation or natural mutation. Feline FcεRα protein homologs of thepresent invention can also be produced using techniques known in the artincluding, but not limited to, direct modifications to the protein ormodifications to the gene encoding the protein using, for example,classic or recombinant nucleic acid techniques to effect random ortargeted mutagenesis.

Isolated feline FcεRα proteins of the present invention have the furthercharacteristic of being encoded by nucleic acid molecules that hybridizeunder stringent hybridization conditions to a gene encoding a felineFcεRα protein. As used herein, stringent hybridization conditions referto standard hybridization conditions under which nucleic acid molecules,including oligonucleotides, are used to identify similar nucleic acidmolecules. Such standard conditions are disclosed, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Labs Press, 1989; Sambrook et al., ibid., is incorporated byreference herein in its entirety. Stringent hybridization conditionstypically permit isolation of nucleic acid molecules having at leastabout 70% nucleic acid sequence identity with the nucleic acid moleculebeing used to probe in the hybridization reaction. Formulae to calculatethe appropriate hybridization and wash conditions to achievehybridization permitting 30% or less mismatch of nucleotides aredisclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138,267-284; Meinkoth et al., ibid., is incorporated by reference herein inits entirety.

As used herein, a feline FcεRα gene includes all nucleic acid sequencesrelated to a natural feline FcεRα gene such as regulatory regions thatcontrol production of the feline FcεRα protein encoded by that gene(such as, but not limited to, transcription, translation orpost-translation control regions) as well as the coding region itself.In one embodiment, a feline FcεRα gene of the present invention includesnucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14 and/or SEQ IDNO:15. Nucleic acid sequence SEQ ID NO:1 represents the deduced sequenceof the coding strand of a complementary DNA (cDNA) nucleic acid moleculedenoted herein as nfelFcεRα₁₀₆₉, the production of which is disclosed inthe Examples. The complement of SEQ ID NO:1 (represented herein by SEQID NO:3) refers to the nucleic acid sequence of the strand complementaryto the strand having SEQ ID NO:1, which can easily be determined bythose skilled in the art. Likewise, a nucleic acid sequence complementof any nucleic acid sequence of the present invention refers to thenucleic acid sequence of the nucleic acid strand that is complementaryto (i.e., can form a complete double helix with) the strand for whichthe sequence is cited.

It should be noted that since nucleic acid sequencing technology is notentirely error-free, SEQ ID NO:1 and SEQ ID NO:3 (as well as othernucleic acid and protein sequences presented herein) represent apparentnucleic acid sequences of certain nucleic acid molecules encoding felineFcεRα proteins of the present invention.

In another embodiment, a feline FcεRα gene can be an allelic variantthat includes a similar but not identical sequence to SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:14, SEQ ID NO:15 and/or SEQ ID NO:16. An allelicvariant of a feline FcεRα gene is a gene that occurs at essentially thesame locus (or loci) in the genome as the gene including SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:14, SEQ ID NO:15 and/or SEQ ID NO:16, but which, due tonatural variations caused by, for example, mutation or recombination,has a similar but not identical sequence. Allelic variants typicallyencode proteins having similar activity to that of the protein encodedby the gene to which they are being compared. Allelic variants can alsocomprise alterations in the 5' or 3' untranslated regions of the gene(e.g., in regulatory control regions). Allelic variants are well knownto those skilled in the art and would be expected to be found within agiven cat since the genome is diploid and/or among a group of two ormore cats. The present invention also includes variants due tolaboratory manipulation, such as, but not limited to, variants producedduring polymerase chain reaction amplification.

The minimal size of a FcεRα protein homolog of the present invention isa size sufficient to be encoded by a nucleic acid molecule capable offorming a stable hybrid (i.e., hybridize under stringent hybridizationconditions) with the complementary sequence of a nucleic acid moleculeencoding the corresponding natural protein. As such, the size of thenucleic acid molecule encoding such a protein homolog is dependent onnucleic acid composition and percent homology between the nucleic acidmolecule and complementary sequence. It should also be noted that theextent of homology required to form a stable hybrid can vary dependingon whether the homologous sequences are interspersed throughout thenucleic acid molecules or are clustered (i.e., localized) in distinctregions on the nucleic acid molecules. The minimal size of such nucleicacid molecules is typically at least about 12 to about 15 nucleotides inlength if the nucleic acid molecules are GC-rich and at least about 15to about 17 bases in length if they are AT-rich. As such, the minimalsize of a nucleic acid molecule used to encode a feline FcεRα proteinhomolog of the present invention is from about 12 to about 18nucleotides in length. Thus, the minimal size of a feline FcεRα proteinhomolog of the present invention is from about 4 to about 6 amino acidsin length. There is no limit, other than a practical limit, on themaximal size of such a nucleic acid molecule in that the nucleic acidmolecule can include a portion of a gene, an entire gene, multiplegenes, or portions thereof. The preferred size of a protein encoded by anucleic acid molecule of the present invention depends on whether afull-length, fusion, multivalent, or functional portion of such aprotein is desired. Preferably, the preferred size of a protein encodedby a nucleic acid molecule of the present invention is a portion of theprotein that binds to IgE which is about 30 amino acids, more preferablyabout 35 amino acids and even more preferably about 44 amino acids inlength.

As used herein, a feline refers to any member of the cat family,including domestic cats, wild cats and zoo cats. Examples of cats fromwhich to isolate feline FcεRα proteins of the present invention(including isolation of the natural protein or production of the proteinby recombinant or synthetic techniques) include, but are not limited to,domestic cats, lions, tigers, leopards, panthers, cougars, bobcats,lynx, jaguars, cheetahs, and servals, with domestic cats being morepreferred and Felis domesticus cats being even more preferred.

Suitable cat cells from which to isolate a feline FcεRα protein of thepresent invention include cells that have FcεR proteins. Preferred catcells from which to obtain a feline FcεRα protein of the presentinvention include basophil cells, mast cells, mastocytoma cells,dendritic cells, B lymphocytes, macrophages, eosinophils, and/ormonocytes. A feline FcεRα of the present invention is preferablyobtained from mastocytoma cells, mast cells or basophil cells.

The present invention also includes mimetopes of feline FcεRα proteinsof the present invention. As used herein, a mimetope of a feline FcεRαprotein of the present invention refers to any compound that is able tomimic the activity of such a feline FcεRα protein (e.g., ability to bindto IgE), often because the mimetope has a structure that mimics thefeline FcεRα: protein. It is to be noted, however, that the mimetopeneed not have a structure similar to a feline FcεRα protein as long asthe mimetope functionally mimics the protein. Mimetopes can be, but arenot limited to: peptides that have been modified to decrease theirsusceptibility to degradation; anti-idiotypic and/or catalyticantibodies, or fragments thereof; non-proteinaceous immunogenic portionsof an isolated protein (e.g., carbohydrate structures); synthetic ornatural organic or inorganic molecules, including nucleic acids; and/orany other peptidomimetic compounds. Mimetopes of the present inventioncan be designed using computer-generated structures of feline FcεRαproteins of the present invention. Mimetopes can also be obtained bygenerating random samples of molecules, such as oligonucleotides,peptides or other organic molecules, and screening such samples byaffinity chromatography techniques using the corresponding bindingpartner, (e.g., a feline IgE Fc domain or anti-feline FcεRα antibody). Amimetope can also be obtained by, for example, rational drug design. Ina rational drug design procedure, the three-dimensional structure of acompound of the present invention can be analyzed by, for example,nuclear magnetic resonance (NMR) or x-ray crystallography. Thethree-dimensional structure can then be used to predict structures ofpotential mimetopes by, for example, computer modeling. The predictedmimetope structures can then be produced by, for example, chemicalsynthesis, recombinant DNA technology, or by isolating a mimetope from anatural source. Specific examples of feline FcεRα mimetopes includeanti-idiotypic antibodies, oligonucleotides produced using Selex™technology, peptides identified by random screening of peptide librariesand proteins identified by phage display technology. A preferredmimetope is a peptidomimetic compound that is structurally and/orfunctionally similar to a feline FcεRα protein of the present invention,particularly to the IgE Fc domain binding site of the felineFcεRαprotein. As used herein, the Fc domain of an antibody refers to theportion of an immunoglobulin that has Fc receptor binding effectorfunction. Typically, the Fc domain of an IgE comprises the CH2 and CH3domains of the heavy chain constant region.

According to the present invention, a feline FcεRα molecule of thepresent invention refers to: a feline FcεRα protein, in particular asoluble feline FcεRα protein; a feline FcεRα homolog; a feline FcεRαmimetope; a feline FcεRα substrate analog; or a feline FcεRα peptide.Preferably, a feline FcεRα molecule binds to IgE.

One embodiment of a feline FcεRα protein of the present invention is afusion protein that includes a feline FcεRα protein-containing domainattached to one or more fusion segments. Suitable fusion segments foruse with the present invention include, but are not limited to, segmentsthat can: enhance a protein's stability; act as an immunopotentiator toenhance an immune response against a feline FcεRα protein; and/or assistpurification of a feline FcεRα protein (e.g., by affinitychromatography). A suitable fusion segment can be a domain of any sizethat has the desired function (e.g., imparts increased stability,imparts increased immunogenicity to a protein, and/or simplifiespurification of a protein). Fusion segments can be joined to aminoand/or carboxyl termini of the feline FcεRα-containing domain of theprotein and can be susceptible to cleavage in order to enablestraight-forward recovery of a feline FcεRα protein. Fusion proteins arepreferably produced by culturing a recombinant cell transformed with afusion nucleic acid molecule that encodes a protein including the fusionsegment attached to either the carboxyl and/or amino terminal end of afeline FcεRα-containing domain. Preferred fusion segments include ametal binding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptoror complement protein antibody-binding domains); a sugar binding domain(e.g., a maltose binding domain); a "tag" domain (e.g., at least aportion of β-galactosidase, a strep tag peptide, other domains that canbe purified using compounds that bind to the domain, such as monoclonalantibodies); and/or a linker and enzyme domain (e.g., alkalinephosphatase domain connected to a feline FcεRα protein by a linker).More preferred fusion segments include metal binding domains, such as apoly-histidine segment; a maltose binding domain; a strep tag peptide,such as that available from Biometra in Tampa, Fla.; and a phage T7 S10peptide.

A preferred feline FcεRα protein of the present invention is encoded bya nucleic acid molecule that hybridizes under stringent hybridizationconditions with at least one of the following nucleic acid molecules:nfelFc.sub.ε Rα₁₀₆₉, nfelFc.sub.ε Rα₇₈₉,nfelFc.sub.ε Rα₇₁₄, nfelFc.sub.εRα₅₉₇,nfelFc.sub.ε Rα₅₂₂. Preferably, the feline FcεRα protein binds toIgE. A further preferred isolated protein is encoded by a nucleic acidmolecule that hybridizes under stringent hybridization conditions with anucleic acid molecule having nucleic acid sequence SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:15 and SEQ ID NO:16.

Translation of SEQ ID NO:1 suggests that nucleic acid moleculenfelFc.sub.ε Rα₁₀₆₉ encodes a full-length feline protein of about 263amino acids, referred to herein as PfelFc.sub.ε Rα₂₆₃, represented bySEQ ID NO:2, assuming an open reading frame having an initiation (start)codon spanning from about nucleotide 65 through about nucleotide 67 ofSEQ ID NO:1 and a termination (stop) codon spanning from aboutnucleotide 854 through about nucleotide 856 of SEQ ID NO:1. The codingregion encoding PfelFc.sub.ε Rα₂₆₃ is represented by nucleic acidmolecule nfelFc.sub.ε Rα₇₈₉, having a coding strand with the nucleicacid sequence represented by SEQ ID NO:4 and a complementary strand withthe nucleic acid sequence represented by SEQ ID NO:5. Analysis of SEQ IDNO:2 suggests the presence of a signal peptide encoded by a stretch ofamino acids spanning from about amino acid 1 through about amino acid25. The proposed mature protein, denoted herein as PfelFc.sub.ε Rα₂₃₈,contains about 238 amino acids which is represented herein as SEQ IDNO:7. PfelFc.sub.ε Rα₂₃₈ is encoded by nucleic acid moleculenfelFc.sub.ε Rα₇₁₄, having a coding strand with the nucleic acidsequence represented by SEQ ID NO:6 and a complementary strand with thenucleic acid sequence represented by SEQ ID NO:8. The amino acidsequence of PfelFc.sub.ε Rα₂₃₈ (i.e. SEQ ID NO:7) predicts thatPfelFc.sub.ε Rα₂₃₈ has an estimated molecular weight of about 30.2 kD,an estimated pIof about 9.51.

Comparison of amino acid sequence SEQ ID NO:2 (i.e., the amino acidsequence of PfelFc.sub.ε Rα₂₆₃) with amino acid sequences reported inGenBank™ indicates that SEQ ID NO:2 showed the most homology, i.e.,about 54% identity, with a Fc epsilon receptor alpha chain protein ofHomo Sapiens (GenBank accession number J03605).

More preferred feline FcεRα proteins of the present invention includeproteins comprising amino acid sequences that are at least about 60%,preferably at least about 65%, more preferably at least about 70%, morepreferably at least about 75%, more preferably at least about 80% andeven more preferably at least about 85%, identical to amino acidsequence SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12 and/or SEQ ID NO:13.

More preferred feline FcεRα proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising at least aportion of nfelFc.sub.ε Rα₁₀₆₉, nfelFc.sub.ε Rα₇₈₉,nfelFc.sub.ε Rα₇₁₄,nfelFc.sub.ε Rα₅₉₇ and nfelFc.sub.ε Rα₅₂₂, or of allelic variants ofsuch nucleic acid molecules, the portion being capable of binding toIgE. More preferred is a feline FcεRα protein encoded by nfelFc.sub.εRα₁₀₆₉, nfelFc.sub.ε Rα₇₈₉,nfelFc.sub.ε Rα₇₁₄, nfelFc.sub.ε Rα₅₉₇ andnfelFc.sub.ε Rα₅₂₂, or by an allelic variant of such nucleic acidmolecules. Particularly preferred feline FcεRα proteins are PfelFc.sub.εRα₂₃₈, PfelFc.sub.ε Rα₂₆₃, PfelFc.sub.ε Rα₁₉₉ and PfelFc.sub.ε Rα₁₇₄.

In one embodiment, a preferred feline FcεRα A protein of the presentinvention is encoded by at least a portion of SEQ ID NO:1, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:11 and/or SEQ ID NO:14, and, as such, has anamino acid sequence that includes at least a portion of SEQ ID NO:2, SEQID NO:7, SEQ ID NO:12 and/or SEQ ID NO:13.

Also preferred is a feline FcεRα A protein encoded by an allelic variantof a nucleic acid molecule comprising at least a portion of SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:11 and/or SEQ ID NO:14. Particularlypreferred feline FcεRα proteins of the present invention include SEQ IDNO:2, SEQ ID NO:7, SEQ ID NO:12 and SEQ ID NO:13 (including, but notlimited to, the proteins consisting of such sequences, fusion proteinsand multivalent proteins) and proteins encoded by allelic variants ofnucleic acid molecules that encode SEQ ID NO:2, SEQ ID NO:7, SEQ IDNO:12 and/or SEQ ID NO:13.

Another embodiment of the present invention is an isolated nucleic acidmolecule that hybridizes under stringent hybridization conditions with afeline FcεRα gene. The identifying characteristics of such a gene areheretofore described. A nucleic acid molecule of the present inventioncan include an isolated natural feline FcεRα gene or a homolog thereof,the latter of which is described in more detail below. A nucleic acidmolecule of the present invention can include one or more regulatoryregions, full-length or partial coding regions, or combinations thereof.The minimal size of a nucleic acid molecule of the present invention isthe minimal size that can form a stable hybrid with a feline FcεRα geneunder stringent hybridization conditions.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,"isolated" does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated feline FcεRα nucleic acidmolecule of the present invention can be isolated from its naturalsource or can be produced using recombinant DNA technology (e.g.,polymerase chain reaction (PCR) amplification, cloning) or chemicalsynthesis. Isolated feline FcεRα nucleic acid molecules can include, forexample, natural allelic variants and nucleic acid molecules modified bynucleotide insertions, deletions, substitutions, and/or inversions in amanner such that the modifications do not substantially interfere withthe nucleic acid molecule's ability to encode a feline FcεRα protein ofthe present invention or to form stable hybrids under stringentconditions with natural gene isolates.

A feline FcεRα nucleic acid molecule homolog can be produced using anumber of methods known to those skilled in the art (see, for example,Sambrook et al., ibid.). For example, nucleic acid molecules can bemodified using a variety of techniques including, but not limited to,classic mutagenesis and recombinant DNA techniques (e.g., site-directedmutagenesis, chemical treatment, restriction enzyme cleavage, ligationof nucleic acid fragments and/or PCR amplification), synthesis ofoligonucleotide mixtures and ligation of mixture groups to "build" amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologs can be selected by hybridization with a feline FcεRαgene or by screening for function of a protein encoded by the nucleicacid molecule (e.g., ability of a feline FcεRα protein to bind IgE).

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one feline FcεRα protein ofthe present invention, examples of such proteins being disclosed herein.Although the phrase "nucleic acid molecule" primarily refers to thephysical nucleic acid molecule and the phrase "nucleic acid sequence"primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a feline FcεRα protein.

One embodiment of the present invention is a feline FcεRα nucleic acidmolecule that hybridizes under stringent hybridization conditions withnucleic acid molecule nfelFc.sub.ε Rα₁₀₆₉ and preferably with a nucleicacid molecule having nucleic acid sequence SEQ ID NO:1 and/or SEQ IDNO:3.

Comparison of nucleic acid sequence SEQ ID NO:1 (i.e., the nucleic acidsequence of the coding strand of nfelFc.sub.ε Rα₁₀₆₉) with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:1 showed the mosthomology, i.e., about 77% identity a canine Fc epsilon receptor alphachain gene.

Preferred feline FcεRα nucleic acid molecules include nucleic acidmolecules having a nucleic acid sequence that is at least about 80%,preferably at least about 85%, more preferably at least about 90%, andeven more preferably at least about 95% identical to nucleic acidsequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:15 and/or SEQID NO:16.

Another preferred nucleic acid molecule of the present inventionincludes at least a portion of nucleic acid sequence SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:15 and/or SEQ ID NO:16, that is capable ofhybridizing to a feline FcεRα gene of the present invention, as well asallelic variants thereof. A more preferred nucleic acid moleculeincludes the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14,SEQ ID NO:15 and/or SEQ ID NO:16, as well as allelic variants of such anucleic acid molecule. Such nucleic acid molecules can includenucleotides in addition to those included in the SEQ ID NOs, such as,but not limited to, a full-length gene, a full-length coding region, anucleic acid molecule encoding a fusion protein, or a nucleic acidmolecule encoding a multivalent protective compound. Particularlypreferred nucleic acid molecules include nfelFc.sub.ε Rα₁₀₆₉,nfelFc.sub.ε Rα₇₈₉, nfelFc.sub.ε Rα₇₁₄, nfelFc.sub.ε Rα₅₉₇ andnfelFc.sub.ε Rα₅₂₂.

The present invention also includes a nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:2, SEQ ID NO:7, SEQ IDNO:12 and SEQ ID NO:13, including nucleic acid molecules that have beenmodified to accommodate codon usage properties of the cells in whichsuch nucleic acid molecules are to be expressed.

Knowing the nucleic acid sequences of certain feline FcεRα nucleic acidmolecules of the present invention allows one skilled in the art to, forexample, (a) make copies of those nucleic acid molecules, (b) obtainnucleic acid molecules including at least a portion of such nucleic acidmolecules (e.g., nucleic acid molecules including full-length genes,full-length coding regions, regulatory control sequences, truncatedcoding regions), and (c) obtain feline FcεRα nucleic acid molecules fromother cats. Such nucleic acid molecules can be obtained in a variety ofways including screening appropriate expression libraries withantibodies of the present invention; traditional cloning techniquesusing oligonucleotide probes of the present invention to screenappropriate libraries or DNA; and PCR amplification of appropriatelibraries or DNA using oligonucleotide primers of the present invention.Preferred libraries to screen or from which to amplify nucleic acidmolecule include feline basophil cell, mast cell, mastocytoma cell,dendritic cell, B lymphocyte, macrophage, eosinophil, and/or monocytecDNA libraries as well as genomic DNA libraries. Similarly, preferredDNA sources to screen or from which to amplify nucleic acid moleculesinclude feline basophil cells, mast cells, mastocytoma cells, dendriticcells, B lymphocytes, macrophages, eosinophils, and/or monocytes cDNAand genomic DNA. Techniques to clone and amplify genes are disclosed,for example, in Sambrook et al., ibid.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingfeline FcεRα genes or other feline FcεRα nucleic acid molecules.Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. Minimal size characteristics are disclosedherein. The present invention includes oligonucleotides that can be usedas, for example, probes to identify nucleic acid molecules, primers toproduce nucleic acid molecules or therapeutic reagents to inhibit felineFcεRα protein production or activity (e.g., as antisense-, triplexformation-, ribozyme- and/or RNA drug-based reagents). The presentinvention also includes the use of such oligonucleotides to protectanimals from disease using one or more of such technologies. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto an animal using techniques known to those skilled in the art.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulation of felineFcεRα nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, endoparasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells and morepreferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, insect and mammaliancells, such as, but not limited to, tac, lac, trp, trc, oxy-pro,omp/lpp, rrnB, bacteriophage lambda (such as lambda p_(L) and lambdap_(R) and fusions that include such promoters), bacteriophage T7, T7lac,bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoters (such as Sindbis virus subgenomic promoters),antibiotic resistance gene, baculovirus, Heliothis zea insect virus,vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus,adenovirus, cytomegalovirus (such as intermediate early promoters),simian virus 40, retrovirus, actin, retroviral long terminal repeat,Rous sarcoma virus, heat shock, phosphate and nitrate transcriptioncontrol sequences as well as other sequences capable of controlling geneexpression in prokaryotic or eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters (e.g., promotersinducible by interferons or interleukins). Transcription controlsequences of the present invention can also include naturally occurringtranscription control sequences naturally associated with cats.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include nfelFc.sub.ε Rα₁₀₆₉,nfelFc.sub.ε Rα₇₈₉, nfelFc.sub.ε Rα₇₁₄, nfelFc.sub.ε Rα₅₉₇ andnfelFc.sub.ε Rα₅₂₂. A particularly preferred recombinant molecule of thepresent invention includes pVL-nfelFc.sub.ε Rα₅₉₇, the production ofwhich are described in the Examples section.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed feline FcεRα protein of the present invention to besecreted from the cell that produces the protein and/or (b) containfusion sequences which lead to the expression of nucleic acid moleculesof the present invention as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionof a protein of the present invention. Preferred signal segmentsinclude, but are not limited to, tissue plasminogen activator (t-PA),interferon, interleukin, growth hormone, histocompatibility and viralenvelope glycoprotein signal segments, as well as natural signalsegments. Suitable fusion segments encoded by fusion segment nucleicacids are disclosed herein. In addition, a nucleic acid molecule of thepresent invention can be joined to a fusion segment that directs theencoded protein to the proteosome, such as a ubiquitin fusion segment.Recombinant molecules may also include intervening and/or untranslatedsequences surrounding and/or within the nucleic acid sequences ofnucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a cell include feline FcεRα nucleic acid molecules disclosedherein. Particularly preferred nucleic acid molecules with which totransform a cell include nfelFc.sub.ε Rα₁₀₆₉, nfelFc.sub.ε Rα₇₈₉,nfelFc.sub.ε Rα₇₁₄, nfelFc.sub.ε Rα₅₉₇ and nfelFc.sub.ε Rα₅₂₂.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing feline FcεRα proteins of the presentinvention or can be capable of producing such proteins after beingtransformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal (including yeast), other insect, other animal andplant cells. Preferred host cells include bacterial, mycobacterial,yeast, parasite, insect and mammalian cells. More preferred host cellsinclude Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces,Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells,MDCK cells (normal dog kidney cell line for canine herpesviruscultivation), CRFK cells (normal cat kidney cell line for felineherpesvirus cultivation), CV-1 cells (African monkey kidney cell lineused, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains such as UK-1 _(x) 3987 andSR-11 _(x) 4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCKcells; CRFK cells; CV-1 cells; COS cells; Vero cells; andnon-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).Additional appropriate mammalian cell hosts include other kidney celllines, other fibroblast cell lines (e.g., human, murine or chickenembryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells. In oneembodiment, the proteins may be expressed as heterologous proteins inmyeloma cell lines employing immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences. The phrase operatively linked refers to insertion of anucleic acid molecule into an expression vector in a manner such thatthe molecule is able to be expressed when transformed into a host cell.

A recombinant molecule of the present invention is a molecule that caninclude at least one of any nucleic acid molecule heretofore describedoperatively linked to at least one of any transcription control sequencecapable of effectively regulating expression of the nucleic acidmolecule(s) in the cell to be transformed, examples of which aredisclosed herein. A particularly preferred recombinant molecule includespVL-nfelFc.sub.ε Rα₅₉₇.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transformcells are disclosed herein. A particularly preferred recombinant cellincludes S. frugiperda:pVL-nfelFc.sub.ε Rα₅₉₇. Details regarding theproduction of this recombinant cell is disclosed herein.

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgamo sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant enzyme productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing nucleic acid molecules encoding such a protein.

Isolated feline FcεRα proteins of the present invention can be producedin a variety of ways, including production and recovery of naturalproteins, production and recovery of recombinant proteins, and chemicalsynthesis of the proteins. In one embodiment, an isolated protein of thepresent invention is produced by culturing a cell capable of expressingthe protein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell of the present invention. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce a felineFcεRα protein of the present invention. Such a medium typicallycomprises an aqueous medium having assimilable carbon, nitrogen andphosphate sources, and appropriate salts, minerals, metals and othernutrients, such as vitamins. Cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes, and petri plates. Culturing can be carried outat a temperature, pH and oxygen content appropriate for a recombinantcell. Such culturing conditions are within the expertise of one ofordinary skill in the art. Examples of suitable conditions are includedin the Examples section.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase "recovering the protein", as well as similarphrases, refers to collecting the whole fermentation medium containingthe protein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved in"substantially pure" form. As used herein, "substantially pure" refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to a feline FcεRαprotein of the present invention or a mimetope thereof (i.e., anti-feline FcεRα antibodies). As used herein, the term "selectively bindsto" a feline FcεRα protein refers to the ability of antibodies of thepresent invention to preferentially bind to specified proteins andmimetopes thereof of the present invention. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example,Sambrook et al., ibid. An anti- feline FcεRα antibody preferablyselectively binds to a feline FcεRα protein in such a way as to reducethe activity of that protein.

Isolated antibodies of the present invention can include antibodies in abodily fluid (such as, but not limited to, serum), or antibodies thathave been purified to varying degrees. Antibodies of the presentinvention can be polyclonal or monoclonal. Functional equivalents ofsuch antibodies, such as antibody fragments and genetically-engineeredantibodies (including single chain antibodies or chimeric antibodiesthat can bind to more than one epitope) are also included in the presentinvention.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope thereof of the present invention to producethe antibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce feline FcεRα proteins ofthe present invention. Antibodies raised against defined proteins ormimetopes can be advantageous because such antibodies are notsubstantially contaminated with antibodies against other substances thatmight otherwise cause interference in a diagnostic assay or side effectsif used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as tools to detect Fc epsilon receptor in thepresence or absence of IgE and/or (b) as tools to screen expressionlibraries and/or to recover desired proteins of the present inventionfrom a mixture of proteins and other contaminants. Furthermore,antibodies of the present invention can be used to target cytotoxicagents to cells having Fc epsilon receptors such as those disclosedherein in order to directly kill such cells. Targeting can beaccomplished by conjugating (i.e., stably joining) such antibodies tothe cytotoxic agents using techniques known to those skilled in the art.Suitable cytotoxic agents are known to those skilled in the art.Antibodies of the present invention, including FcεRα-binding portionsthereof, can also be used, for example, to inhibit binding of IgE to Fcepsilon receptors, to produce anti-feline FcεRα idiotypic antibodies, topurify cells having feline FcεRα proteins, to stimulate intracellularsignal transduction through a feline FcεRα. and to identify cells havingfeline FcεRα proteins.

A feline FcεRα molecule of the present invention can include chimericmolecules comprising a portion of a feline FcεRα molecule that binds toan IgE and a second molecule that enables the chimeric molecule to bebound to a substrate in such a manner that the FcεRα molecule portionbinds to IgE in essentially the same manner as a FcεRα molecule that isnot bound to a substrate. An example of a suitable second moleculeincludes a portion of an immunoglobulin molecule or another ligand thathas a suitable binding partner that can be immobilized on a substrate,e.g., biotin and avidin, or a metal-binding protein and a metal (e.g.,His), or a sugar-binding protein and a sugar (e.g., maltose).

A feline FcεRα molecule of the present invention can be contained in aformulation, herein referred to as a FcεRα molecule formulation. Forexample, a feline FcεRα molecule can be combined with a buffer in whichthe feline FcεRα molecule is solubilized, and/or with a carrier.Suitable buffers and carriers are known to those skilled in the art.Examples of suitable buffers include any buffer in which a feline FcεRαmolecule can function to selectively bind to IgE, such as, but notlimited to, phosphate buffered saline, water, saline, phosphate buffer,bicarbonate buffer, HEPES buffer(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline),TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer(Tris-acetate-EDTA). Examples of carriers include, but are not limitedto, polymeric matrices, toxoids, and serum albumins, such as bovineserum albumin. Carriers can be mixed with feline FcεRα molecules orconjugated (i.e., attached) to feline FcεRα molecules in such a manneras to not substantially interfere with the ability of the feline FcεRαmolecules to selectively bind to IgE.

A feline FcεRα protein of the present invention can be bound to thesurface of a cell comprising the feline FcεRα protein. A preferredfeline FcεRα protein-bearing cell includes a recombinant cell comprisinga nucleic acid molecule encoding a feline FcεRα protein of the presentinvention. A more preferred recombinant cell of the present inventioncomprises a nucleic acid molecule that encodes at least one of thefollowing proteins: PfelFc.sub.ε Rα₂₃₈ and PfelFc.sub.ε Rα₂₆₃. An evenmore preferred recombinant cell comprises a nucleic acid moleculeincluding nfelFc.sub.ε Rα₁₀₆₉, nfelFc.sub.ε Rα₇₈₉ and nfelFc.sub.ε Rα₇₁₄with a recombinant cell comprising a nucleic acid molecule comprising anucleic acid sequence including SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:6,or a nucleic acid molecule comprising an allelic variant of a nucleicacid molecule comprising SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:6, beingeven more preferred.

In addition, a feline FcεRα molecule formulation of the presentinvention can include not only a feline FcεRα molecule but also one ormore additional antigens or antibodies useful in detecting IgE. As usedherein, an antigen refers to any molecule capable of being selectivelybound by an antibody. As used herein, selective binding of a firstmolecule to a second molecule refers to the ability of the firstmolecule to preferentially bind (e.g., having higher affinity higheravidity) to the second molecule when compared to the ability of a firstmolecule to bind to a third molecule. The first molecule need notnecessarily be the natural ligand of the second molecule. Examples ofsuch antibodies include, but are not limited to, antibodies that bindselectively to the constant region of an IgE heavy (i.e., anti-IgEisotype antibody) or antibodies that bind selectively to an IgE having aspecific antigen specificity (i.e., anti-IgE idiotypic antibody).Suitable anti-IgE antibodies for use in a formulation of the presentinvention are not capable of cross-linking two or more IgE antibodies.Preferred anti-IgE antibodies include Fab fragments of the antibodies(as defined in Janeway et al., ibid.). Examples of such antigens includeany antigen known to induce the production of IgE. Preferred antigensinclude allergens and parasite antigens. Allergens of the presentinvention are preferably derived from fungi, trees, weeds, shrubs,grasses, wheat, corn, soybeans, rice, eggs, milk, cheese, bovines (orcattle), poultry, swine, cats, sheep, yeast, fleas, flies, mosquitos,mites, midges, biting gnats, lice, bees, wasps, ants, true bugs orticks. A suitable flea allergen includes an allergen derived from aflea, in particular flea saliva antigen. A preferred flea allergenincludes a flea saliva antigen. Preferred flea saliva antigens includeantigens such as those disclosed in PCT Patent Publication No. WO96/11271, published Apr. 18, 1996, by Frank et al. (this publication isincorporated by reference herein in its entirety), with flea salivaproducts and flea saliva proteins being particularly preferred.According to the present invention, a flea saliva protein includes aprotein produced by recombinant DNA methods, as well as proteinsisolated by other methods disclosed in PCT Patent Publication No. WO96/11271.

Preferred general allergens include those derived from grass, MeadowFescue, curly dock, plantain, Mexican firebush, lamb's quarters,pigweed, ragweed, sage, elm, cocklebur, box elder, walnut, cottonwood,ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides,Alternaria, Aspergillus, Cladoosporium, Fusarium, Helminthosporium,Mucor, Penicillium, Pullularia, Rhizopus and/or Tricophyton. Morepreferred general allergens include those derived from Johnson grass,Kentucky blue grass, meadow fescue, orchard grass, perennial rye grass,red top grass, timothy grass, Bermuda grass, brome grass, curly dock,English plantain, Mexican firebush, lamb's quarters, rough pigweed shortragweed, wormwood sage, American elm, common cocklebur, box elder, blackwalnut, eastern cottonwood, green ash, river birch, red cedar, red oak,red mulberry, cockroach, Dermataphagoides farinae, Alternaria alternata,Aspergillus fumnigatus, Cladosporium herbarum, Fusarium vasinfectum,Helmninthosporium sativum, Mucor recemosus, Penicillium notatum,Pullularia pullulans, Rhizopus nigricans and/or Tricophyton spp.Preferred parasite antigens include, but are not limited to, helminthantigens, in particular heartworm antigens, such as Di33 (described inU.S. patent application Ser. No. 08/715,628, filed Sep. 18, 1996, byGrieve et al., which is incorporated by reference herein in itsentirety). The term "derived from" refers to a natural allergen of suchplants or organisms (i.e., an allergen directly isolated from suchplants or organisms), as well as, non-natural allergens of such plantsor organisms that posses at least one epitope capable of eliciting animmune response against an allergen (e.g., produced using recombinantDNA technology or by chemical synthesis).

One embodiment of the present invention is a method to detect IgE whichincludes the steps of: (a) contacting an isolated feline FcεRα moleculewith a putative IgE-containing composition under conditions suitable forformation of a feline FcεRα molecule:IgE complex; and (b) detecting thepresence of IgE by detecting the feline FcεRα molecule:IgE complex.Presence of such a feline FcεRα molecule:IgE complex indicates that theanimal is producing IgE. Preferred IgE to detect using a feline FcεRαmolecule include feline IgE, canine IgE, equine IgE and human IgE, withfeline IgE being particularly preferred. The present method can furtherinclude the step of determining whether an IgE complexed with a felineFcεRα protein is heat labile. Preferably, a heat labile IgE isdetermined by incubating an IgE at about 56° C. for about 3 or about 4hours. Without being bound by theory, the inventors believe that heatlabile forms of IgE bind to certain allergens and non-heat labile formsof IgE bind to other types of allergens. As such, detection of heatlabile IgE compared with non-heat labile IgE can be used to discriminatebetween allergen sensitivities. For example, the inventors believe thatIgE antibodies that bind to certain flea allergens and heartwormallergens are heat labile while IgE antibodies that bind to certainplant allergens are not heat labile. Thus, the presence of non-heatlabile IgE may indicate that an animal is sensitive to certain plantallergens but not to certain flea or heartworm allergens. Moreover, theinventors believe that identification of heat labile IgE and non-heatlabile IgE using a feline FcεRα protein suggests the presence ofdifferent sub-populations of IgE that may or may not have substantiallysimilar structures to known IgE antibodies. As such, a feline FcεRαprotein of the present invention may be useful for detecting moleculesbound by the feline FcεRα protein but not identical to a known IgE.

As used herein, canine refers to any member of the dog family, includingdomestic dogs, wild dogs and zoo dogs. Examples of dogs include, but arenot limited to, domestic dogs, wild dogs, foxes, wolves, jackals andcoyotes. As used herein, equine refers to any member of the horsefamily, including horses, donkeys, mules and zebras.

As used herein, the term "contacting" refers to combining or mixing, inthis case a putative IgE-containing composition with a feline FcεRαmolecule. Formation of a complex between a feline FcεRα molecule and anIgE refers to the ability of the feline FcεRα molecule to selectivelybind to the IgE in order to form a stable complex that can be measured(i.e., detected). As used herein, the term selectively binds to an IgErefers to the ability of a feline FcεRα molecule of the presentinvention to preferentially bind to IgE, without being able tosubstantially bind to other antibody isotypes. Binding between a felineFcεRα molecule and an IgE is effected under conditions suitable to forma complex; such conditions (e.g., appropriate concentrations, buffers,temperatures, reaction times) as well as methods to optimize suchconditions are known to those skilled in the art, and examples aredisclosed herein. Examples of complex formation conditions are alsodisclosed in, for example, in Sambrook et al., ibid.

As used herein, the term "detecting complex formation" refers todetermining if any complex is formed, i.e., assaying for the presence(i.e., existence) of a complex. If complexes are formed, the amount ofcomplexes formed can, but need not be, determined. Complex formation, orselective binding, between feline FcεRα molecule and any IgE in thecomposition can be measured (i.e., detected, determined) using a varietyof methods standard in the art (see, for example, Sambrook et al.ibid.), examples of which are disclosed herein.

In one embodiment, a putative IgE-containing composition of the presentmethod includes a biological sample from an animal. A suitablebiological sample includes, but is not limited to, a bodily fluidcomposition or a cellular composition. A bodily fluid refers to anyfluid that can be collected (i.e., obtained) from an animal, examples ofwhich include, but are not limited to, blood, serum, plasma, urine,tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasalsecretions, milk and feces. Such a composition of the present methodcan, but need not be, pretreated to remove at least some of the non-IgEisotypes of immunoglobulin and/or other proteins, such as albumin,present in the fluid. Such removal can include, but is not limited to,contacting the bodily fluid with a material, such as Protein G, toremove IgG antibodies and/or affinity purifying IgE antibodies fromother components of the body fluid by exposing the fluid to, forexample, Concanavalin A. In another embodiment, a composition includescollected bodily fluid that is pretreated to concentrate immunoglobulincontained in the fluid. For example, immunoglobulin contained in abodily fluid can be precipitated from other proteins using ammoniumsulfate. A preferred composition of the present method is serum.

In another embodiment, a IgE-containing composition of the presentmethod includes a cell that produces IgE. Such a cell can have IgE boundto the surface of the cell and/or can secrete IgE. An example of such acell includes myeloma cells. IgE can be bound to the surface of a celleither directly to the membrane of the cell or bound to a molecule(e.g., an antigen) bound to the surface of the cell.

A complex can be detected in a variety of ways including, but notlimited to use of one or more of the following assays: an enzyme-linkedimmunoassay, a radioimmunoassay, a fluorescence immunoassay, achemiluminescent assay, a lateral flow assay, an agglutination assay, aparticulate-based assay (e.g., using particulates such as, but notlimited to, magnetic particles or plastic polymers, such as latex orpolystyrene beads), an immunoprecipitation assay, a BioCore™ assay(e.g., using colloidal gold) and an immunoblotting assay (e.g., awestern blot). Such assays are well known to those skilled in the art.Assays can be used to give qualitative or quantitative results dependingon how they are used. Some assays, such as agglutination, particulateseparation, and immunoprecipitation, can be observed visually (e.g.,either by eye or by a machines, such as a densitometer orspectrophotometer) without the need for a detectable marker. In otherassays, conjugation (i.e., attachment) of a detectable marker to thefeline FcεRα molecule or to a reagent that selectively binds to thefeline FcεRα molecule or to the IgE being detected (described in moredetail below) aids in detecting complex formation. Examples ofdetectable markers include, but are not limited to, a radioactive label,an enzyme, a fluorescent label, a chemiluminescent label, a chromophoriclabel or a ligand. A ligand refers to a molecule that binds selectivelyto another molecule. Preferred detectable markers include, but are notlimited to, fluorescein, a radioisotope, a phosphatase (e.g., alkalinephosphatase), biotin, avidin, a peroxidase (e.g., horseradishperoxidase) and biotin-related compounds or avidin-related compounds(e.g., streptavidin or ImmunoPure® NeutrAvidin available from Pierce,Rockford, Ill.). According to the present invention, a detectable markercan be connected to a feline FcεRα molecule using, for example, chemicalconjugation or recombinant DNA technology (e.g., connection of a fusionsegment such as that described herein for a metal binding domain; animmunoglobulin binding; a sugar binding domain; and a "tag" domain).Preferably a carbohydrate group of the feline FcεRα molecule ischemically conjugated to biotin.

In one embodiment, a complex is detected by contacting a putativeIgE-containing composition with a feline FcεRα molecule that isconjugated to a detectable marker. A suitable detectable marker toconjugate to a feline FcεRα molecule includes, but is not limited to, aradioactive label, a fluorescent label, an enzyme label, achemiluminescent label, a chromophoric label or a ligand. A detectablemarker is conjugated to a feline FcεRα molecule in such a manner as notto block the ability of the feline FcεRα molecule to bind to the IgEbeing detected. Preferably, a carbohydrate group of a feline FcεRαmolecule is conjugated to biotin.

In another embodiment, a feline FcεRα molecule:IgE complex is detectedby contacting a putative IgE-containing composition with a feline FcεRαmolecule and then contacting the complex with an indicator molecule.Suitable indicator molecules of the present invention include moleculesthat can bind to either the feline FcεRα molecule or to the IgEantibody. As such, an indicator molecule can comprise, for example, anantigen, an antibody and a lectin, depending upon which portion of thefeline FcεRα molecule:IgE complex is being detected. Preferred indicatormolecules that are antibodies include, for example, anti-IgE antibodiesand anti-feline FcεRα antibodies. Preferred lectins include thoselectins that bind to high-mannose groups. More preferred lectins bind tohigh-mannose groups present on a feline FcεRα protein of the presentinvention produced in insect cells. An indicator molecule itself can beattached to a detectable marker of the present invention. For example,an antibody can be conjugated to biotin, horseradish peroxidase,alkaline phosphatase or fluorescein.

In one preferred embodiment, a feline FcεRα molecule:IgE complex isdetected by contacting the complex with an indicator molecule thatselectively binds to a feline FcεRα molecule of the present invention.Examples of such indicator molecule includes, but are not limited to, anantibody that selectively binds to a feline FcεRα molecule (referred toherein as an anti-feline FcεRα antibody) or a compound that selectivelybinds to a detectable marker conjugated to a feline FcεRα molecule. Afeline FcεRα molecule conjugated to biotin is preferably detected usingstreptavidin, more preferably using ImmunoPure® NeutrAvidin (availablefrom Pierce, Rockford, Ill.).

In another preferred embodiment, a feline FcεRα molecule:Ige complex isdetected by contacting the complex with indicator molecule thatselectively binds to an IgE antibody (referred to herein as an anti-IgEreagent). Examples of such an anti-IgE antibody include, but are notlimited to, a secondary antibody that is an anti-isotype antibody (e.g.,an antibody that selectively binds to the constant region of an IgE), anantibody-binding bacterial surface protein (e.g., Protein A or ProteinG), an antibody-binding cell (e.g., a B cell, a T cell, a natural killercell, a polymorphonuclear leukocyte cell, a monocyte cell or amacrophage cell), an antibody-binding eukaryotic cell surface protein(e.g., a Fc receptor), and an antibody-binding complement protein. Apreferred indicator molecule includes an anti-feline IgE antibody. Asused herein, an anti-IgE antibody includes not only a complete antibodybut also any subunit or portion thereof that is capable of selectivelybinding to an IgE heavy chain constant region. For example, an anti-IgEreagent can include an Fab fragment or a F(ab')₂ fragment, both of whichare described in detail in Janeway et al., in Immunobiology, the ImmuneSystem in Health and Disease, Garland Publishing, Inc., N.Y., 1996(which is incorporated herein by this reference in its entirety).

In one embodiment a complex can be formed and detected in solution. Inanother embodiment, a complex can be formed in which one or more membersof the complex are immobilized on (e.g., coated onto) a substrate.Immobilization techniques are known to those skilled in the art.Suitable substrate materials include, but are not limited to, plastic,glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon,nitrocellulose, and particulate materials such as latex, polystyrene,nylon, nitrocellulose, agarose and magnetic resin. Suitable shapes forsubstrate material include, but are not limited to, a well (e.g.,microtiter dish well), a plate, a dipstick, a bead, a lateral flowapparatus, a membrane, a filter, a tube, a dish, a celluloid-typematrix, a magnetic particle, and other particulates. A particularlypreferred substrate comprises an ELISA plate, a dipstick, aradioimmunoassay plate, agarose beads, plastic beads, latex beads,immunoblot membranes and immunoblot papers. In one embodiment, asubstrate, such as a particulate, can include a detectable marker.

A preferred method to detect IgE is an immunosorbent assay. Animmunoabsorbent assay of the present invention comprises a capturemolecule and an indicator molecule. A capture molecule of the presentinvention binds to an IgE in such a manner that the IgE is immobilizedto a substrate. As such, a capture molecule is preferably immobilized toa substrate of the present invention prior to exposure of the capturemolecule to a putative IgE-containing composition. An indicator moleculeof the present invention detects the presence of an IgE bound to acapture molecule. As such, an indicator molecule preferably is notimmobilized to the same substrate as a capture molecule prior toexposure of the capture molecule to a putative IgE-containingcomposition.

A preferred immunoabsorbent assay method includes a step of either: (a)immobilizing a feline FcεRα molecule on a substrate prior to contactinga feline FcεRα molecule with a putative IgE-containing composition toform a feline FcεRα molecule-immobilized substrate; and (b) binding aputative IgE-containing composition on a substrate prior to contacting afeline FcεRα molecule with a putative IgE-containing composition to forma putative IgE-containing composition-bound substrate. Preferably, thesubstrate includes a non-coated substrate, a feline FcεRαmolecule-immobilized substrate, an antigen-immobilized substrate or ananti-IgE antibody-immobilized substrate.

Both a capture molecule and an indicator molecule of the presentinvention are capable of binding to an IgE. Preferably, a capturemolecule binds to a different region of an IgE than an indicatormolecule, thereby allowing a capture molecule to be bound to an IgE atthe same time as an indicator molecule. The use of a reagent as acapture molecule or an indicator molecule depends upon whether themolecule is immobilized to a substrate when the molecule is exposed toan IgE. For example, a feline FcεRα molecule of the present invention isused as a capture molecule when the feline FcεRα molecule is bound on asubstrate. Alternatively, a feline FcεRα molecule is used as anindicator molecule when the feline FcεRα molecule is not bound on asubstrate. Suitable molecules for use as capture molecules or indicatormolecules include, but are not limited to, a feline FcεRα molecule ofthe present invention, an antigen reagent or an anti-IgE antibodyreagent of the present invention.

An immunoabsorbent assay of the present invention can further compriseone or more layers and/or types of secondary molecules or other bindingmolecules capable of detecting the presence of an indicator molecule.For example, an untagged (i.e., not conjugated to a detectable marker)secondary antibody that selectively binds to an indicator molecule canbe bound to a tagged (i.e., conjugated to a detectable marker) tertiaryantibody that selectively binds to the secondary antibody. Suitablesecondary antibodies, tertiary antibodies and other secondary ortertiary molecules can be selected by those of skill in the art.Preferred secondary molecules of the prescnt invention include anantigen, an anti-IgE idiotypic antibody and an anti-IgE isotypicantibody. Preferred tertiary molecules can be selected by a skilledartisan based upon the characteristics of the secondary molecule. Thesame strategy is applied for subsequent layers.

In one embodiment, a specific antigen is used as a capture molecule bybeing immobilized on a substrate, such as a microtiter dish well or adipstick. Preferred antigens include those disclosed herein. Abiological sample collected from an animal is applied to the substrateand incubated under conditions suitable (i.e., sufficient) to allow forantigen:IgE complex formation bound to the substrate (i.e., IgE in asample binds to an antigen immobilized on a substrate). Excess non-boundmaterial (i.e., material from the biological sample that has not boundto the antigen), if any, is removed from the substrate under conditionsthat retain antigen:IgE complex binding to the substrate. Preferredconditions are generally disclosed in Sambrook et al., ibid. Anindicator molecule that can selectively bind to an IgE bound to theantigen is added to the substrate and incubated to allow formation of acomplex between the indicator molecule and the antigen:IgE complex.Excess indicator molecule is removed, a developing agent is added ifrequired and the substrate is submitted to a detection device foranalysis. A preferred indicator molecule for this embodiment is a felineFcεRα molecule, preferably conjugated to biotin, to a fluorescent labelor to an enzyme label.

In one embodiment, a feline FcεRα molecule is used as a capture moleculeby being immobilized on a substrate, such as a microtiter dish well or adipstick. A biological sample collected from an animal is applied to thesubstrate and incubated under conditions suitable to allow for felineFcεRα molecule:IgE complex formation bound to the substrate. Excessnon-bound material, if any, is removed from the substrate underconditions that retain feline FcεRα molecule:IgE complex binding to thesubstrate. An indicator molecule that can selectively bind to an IgEbound to the feline FcεRα molecule is added to the substrate andincubated to allow formation of a complex between the indicator moleculeand the feline FcεRα molecule:IgE complex. Preferably, the indicatormolecule is conjugated to a detectable marker (preferably to an enzymelabel, to a calorimetric label, to a fluorescent label, to aradioisotope, or to a ligand such as of the biotin or avidin family).Excess indicator molecule is removed, a developing agent is added ifrequired, and the substrate is submitted to a detection device foranalysis. A preferred indicator molecule for this embodiment is anantigen that will bind to IgE in the biological sample or an anti-IgEisotype or idiotype antibody, either preferably being conjugated tofluorescein or biotin.

In one embodiment, an anti-IgE antibody (e.g., isotype or idiotypespecific antibody) is used as a capture molecule by being immobilized ona substrate, such as a microtiter dish well or a dipstick. A biologicalsample collected from an animal is applied to the substrate andincubated under conditions suitable to allow for anti-IgE antibody:IgEcomplex formation bound to the substrate. Excess non-bound material, ifany, is removed from the substrate under conditions that retain anti-IgEantibody:IgE complex binding to the substrate. A feline FcεRα moleculeis added to the substrate and incubated to allow formation of a complexbetween the feline FcεRα molecule and the anti-IgE antibody:IgE complex.Preferably, the feline FcεRα molecule is conjugated to a detectablemarker (preferably to biotin, an enzyme label or a fluorescent label).Excess feline FcεRα molecule is removed, a developing agent is added ifrequired, and the substrate is submitted to a detection device foranalysis.

In one embodiment, an immunosorbent assay of the present invention doesnot utilize a capture molecule. In this embodiment, a biological samplecollected from an animal is applied to a substrate, such as a microtiterdish well or a dipstick, and incubated under conditions suitable toallow for IgE binding to the substrate. Any IgE present in the bodilyfluid is immobilized on the substrate. Excess non-bound material, ifany, is removed from the substrate under conditions that retain IgEbinding to the substrate. A feline FcεRα molecule is added to thesubstrate and incubated to allow formation of a complex between thefeline FcεRα molecule and the IgE. Preferably, the feline FcεRα moleculeis conjugated to a detectable marker (preferably to biotin, an enzymelabel or a fluorescent label). Excess feline FcεRα molecule is removed,a developing agent is added if required, and the substrate is submittedto a detection device for analysis.

Another preferred method to detect IgE is a lateral flow assay, examplesof which are disclosed in U.S. Pat. No. 5,424,193, issued Jun. 13, 1995,by Pronovost et al.; U.S. Pat. No. 5,415,994, issued May 16, 1995, byImrich et al; WO 94/29696, published Dec. 22, 1994, by Miller et al.;and WO 94/01775, published Jan. 20, 1994, by Pawlak et al.; each ofthese patent publications is incorporated by reference herein in itsentirety. In one embodiment, a biological sample is placed in a lateralflow apparatus that includes the following components: (a) a supportstructure defining a flow path; (b) a labeling reagent comprising a beadconjugated to an antigen, the labeling reagent being impregnated withinthe support structure in a labeling zone; and (c) a capture reagentcomprising an IgE-binding composition. Preferred antigens include thosedisclosed herein. The capture reagent is located downstream of thelabeling reagent within a capture zone fluidly connected to the labelingzone in such a manner that the labeling reagent can flow from thelabeling zone into the capture zone. The support structure comprises amaterial that does not impede the flow of the beads from the labelingzone to the capture zone. Suitable materials for use as a supportstructure include ionic (i.e., anionic or cationic) material. Examplesof such a material include, but are not limited to, nitrocellulose (NC),PVDF, carboxymethylcellulose (CM). The support structure defines a flowpath that is lateral and is divided into zones, namely a labeling zoneand a capture zone. The apparatus can further comprise a samplereceiving zone located along the flow path, more preferably upstream ofthe labeling reagent. The flow path in the support structure is createdby contacting a portion of the support structure downstream of thecapture zone, preferably at the end of the flow path, to an absorbentcapable of absorbing excess liquid from the labeling and capture zones.

In this embodiment, the biological sample is applied to the samplereceiving zone which includes a portion of the support structure. Thelabeling zone receives the sample from the sample receiving zone whichis directed downstream by the flow path. The labeling zone comprises thelabeling reagent that binds to IgE. A preferred labeling reagent is anantigen conjugated, either directly or through a linker, to a plasticbead substrate, such as to a latex bead. The substrate also includes adetectable marker, preferably a calorimetric marker. Typically, thelabeling reagent is impregnated to the support structure by drying orlyophilization. The sample structure also comprises a capture zonedownstream of the labeling zone. The capture zone receives labelingreagent from the labeling zone which is directed downstream by the flowpath. The capture zone contains the capture reagent, in this case afeline FcεRα molecule, as disclosed above, that immobilizes the IgEcomplexed to the antigen in the capture zone. The capture reagent ispreferably fixed to the support structure by drying or lyophilizing. Thelabeling reagent accumulates in the capture zone and the accumulation isassessed visually or by an optical detection device.

In another embodiment, a lateral flow apparatus used to detect IgEincludes: (a) a support structure defining a flow path; (b) a labelingreagent comprising a feline FcεRα molecule as described above, thelabeling reagent impregnated within the support structure in a labelingzone; and (c) a capture reagent comprising an antigen, the capturereagent being located downstream of the labeling reagent within acapture zone fluidly connected to the labeling zone in such a mannerthat the labeling reagent can flow from the labeling zone into thecapture zone. The apparatus preferably also includes a sample receivingzone located along the flow path, preferably upstream of the labelingreagent. The apparatus preferably also includes an absorbent located atthe end of the flow path.

One embodiment of the present invention is an inhibition assay in whichthe presence of IgE in a putative IgE-containing composition isdetermined by adding such composition to a feline FcεRα molecule of thepresent invention and an isolated IgE known to bind to the feline FcεRαmolecule. The absence of binding of the feline FcεRα molecule to theknown IgE indicates the presence of IgE in the putative IgE-containingcomposition. The known IgE is preferably conjugated to a detectablemarker.

The present invention also includes kits to detect IgE based on each ofthe disclosed detection methods. One embodiment is a kit to detect IgEcomprising a feline FcεRα protein and a means for detecting an IgE.Suitable and preferred feline FcεRα protein are disclosed herein.Suitable means of detection include compounds disclosed herein that bindto either the feline FcεRα protein or to an IgE. A preferred kit of thepresent invention further comprises a detection means including one ormore antigens disclosed herein, an antibody capable of selectivelybinding to an IgE disclosed herein and/or a compound capable of bindingto a detectable marker conjugated to a feline FcεRα protein (e.g.,avidin, streptavidin and ImmunoPure® NeutrAvidin when the detectablemarker is biotin). Such antigens preferably induce IgE antibodyproduction in animals including canines, felines and/or equines.

A preferred embodiment of a kit of the present invention is a fleaallergen kit comprising a flea allergen such as those disclosed herein.A particularly preferred flea allergen for use with a flea allergen kitincludes a flea saliva product and/or an isolated flea saliva protein.

Another preferred kit of the present invention is a general allergen kitcomprising an allergen common to all regions of the United States and afeline FcεRα protein of the present invention. As used herein, a"general allergen" kit refers to a kit comprising allergens that arefound substantially throughout the United States (i.e., essentially notlimited to certain regions of the United States). A general allergen kitprovides an advantage over regional allergen kits because a single kitcan be used to test an animal located in most geographical locations onthe United States. Suitable and preferred general allergens for use witha general allergen kit of the present invention include those generalallergens disclosed herein.

Another preferred kit of the present invention is a food allergen kitcomprising a food allergen including beef, chicken, pork, a mixture offish, such as cod, halibut or and tuna, egg, milk, Brewer's yeast, wholewheat, corn, soybean, cheese and rice, and a feline FcεRα molecule ofthe present invention. Preferably, the beef, chicken, pork, fish, cornand rice, are cooked.

A preferred kit of the present invention includes those in which theallergen is immobilized on a substrate. If a kit comprises two or moreantigens, the kit can comprise one or more compositions, eachcomposition comprising one antigen. As such, each antigen can be testedseparately. A kit can also contain two or more diagnostic reagents forIgE, additional isolated IgE antigens and/or antibodies as disclosedherein. Particularly preferred are kits used in a lateral flow assayformat. It is within the scope of the present invention that a lateralflow assay kit can include one or more lateral flow assay apparatuses.Multiple lateral flow apparatuses can be attached to each other at oneend of each apparatus, thereby creating a fan-like structure.

In particular, a method and kit of the present invention are useful fordiagnosing abnormal conditions in animals that are associated withchanging levels of IgE. Particularly preferred conditions to diagnoseinclude allergies, parasitic infections and neoplasia. For example, amethod and kit of the present invention are particularly useful fordetecting flea allergy dermatitis (FAD), when such method or kitincludes the use of a flea saliva antigen. FAD is defined as ahypersensitive response to fleabites. Preferably, a putativeIgE-containing composition is obtained from an animal suspected ofhaving FAD. Preferred animals include those disclosed herein, with dogsand cats being more preferred. In addition, methods and kits of thepresent invention are particularly useful for detecting helminthinfection, in particular heartworm infection, when such methods or kitsinclude the use of a helminth antigen, such as Di33. Preferably, aputative IgE-containing composition is obtained from an animal suspectedof having a helminth infection. Preferred animals include thosedisclosed herein, with dogs and cats being more preferred.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is capableof reducing Fc receptor mediated reactions associated with diseasesrelated to biological responses involving Fc receptor function. Atherapeutic composition of the present invention can include: anisolated feline FcεRα protein, or homolog thereof; a mimetope of afeline FcεRα protein; an isolated nucleic acid molecule that hybridizesunder stringent hybridization conditions with a feline FcεRα gene; anisolated antibody that selectively binds to a feline FcεRα protein;and/or an inhibitor that interferes with formation of a complex betweena feline FcεRα protein and IgE.

One embodiment of a therapeutic composition of the present invention isa therapeutic compound comprising a feline FcεRα molecule of the presentinvention, that binds to an IgE. According to the present invention, afeline FcεRα molecule competes for IgE with naturally-occurring Fcepsilon receptors, particularly those on mastocytoma cells, mast cellsor basophils, so that IgE is bound to the administered feline FcεRαmolecule and thus is unable to bind to Fc epsilon receptor on a cell,thereby inhibiting mediation of a biological response. Preferred felineFcεRα molecule for use in a therapeutic composition comprises a felineFcεRα protein, or homolog thereof, as described herein, particularly afragment thereof, which binds to IgE. Feline FcεRα molecules for use ina therapeutic composition can be in a monovalent and/or multivalentform, so long as the feline FcεRα molecule is capable of binding to IgE.A more preferred feline FcεRα molecule for use in a therapeuticcomposition includes a soluble fragment of a feline FcεRα protein. Apreferred feline FcεRα protein is encoded by nfelFc.sub.ε Rα₅₂₂ and aneven more preferred feline FcεRα protein is PfelFc.sub.ε Rα₁₇₄.

Examples of suitable nucleic acid molecules for use in a therapeuticcomposition of the present invention are disclosed herein.

Another embodiment of a therapeutic composition of the present inventioncomprises a therapeutic compound that interferes with the formation of acomplex between feline FcεRα protein and IgE, usually by binding to orotherwise interacting with or otherwise modifying the feline FcεRα Aprotein's IgE binding site. Feline FcεRα protein inhibitors can alsointeract with other regions of the feline FcεRα protein to inhibitfeline FcεRα protein activity, for example, by allosteric interaction.An inhibitor of a feline FcεRα protein can interfere with FcεRα proteinand IgE complex formation by, for example, preventing formation of aFcεRα protein and IgE complex or disrupting an existing FcεRα proteinand IgE complex causing the FcεRα protein and IgE to dissociate. Aninhibitor of a feline FcεRα protein is usually a relatively small.Preferably, a feline FcεRα protein inhibitor of the present invention isidentified by its ability to bind to, or otherwise interact with, afeline FcεRα protein, thereby interfering with the formation of acomplex between a feline FcεRα protein and IgE.

Preferred inhibitors of a feline FcεRα protein of the present inventioninclude, but are not limited to, a substrate analog of a feline FcεRαprotein, a mimetope of a feline FcεRα protein, a soluble (i.e., secretedform of a feline FcεRα protein) portion of a feline FcεRα protein thatbinds to IgE, and other molecules that bind to a feline FcεRα protein(e.g., to an allosteric site) in such a manner that IgE-binding activityof the feline FcεRα protein is inhibited. A feline FcεRα proteinsubstrate analog refers to a compound that interacts with (e.g., bindsto, associates with, modifies) the IgE-binding site of a feline FcεRαprotein. A preferred feline FcεRα protein substrate analog inhibitsIgE-binding activity of a feline FcεRα protein. Feline FcεRα proteinsubstrate analogs can be of any inorganic or organic composition, and,as such, can be, but are not limited to, peptides, nucleic acids, andpeptidomimetic compounds. Feline FcεRα protein substrate analogs can be,but need not be, structurally similar to a feline FcεRα protein'snatural substrate (e.g., IgE) as long as they can interact with theactive site (e.g., IgE-binding site of that feline FcεRα). Feline FcεRαprotein substrate analogs can be designed using computer-generatedstructures of feline FcεRα proteins of the present invention or computerstructures of, for example, the Fc domain of IgE. Substrate analogs canalso be obtained by generating random samples of molecules, such asoligonucleotides, peptides, peptidomimetic compounds, or other inorganicor organic molecules, and screening such samples by affinitychromatography techniques using the corresponding binding partner,(e.g., a feline FcεRα protein or anti-feline FcεRα idiotypic antibody).A preferred feline FcεRα protein substrate analog is a peptidomimeticcompound (i.e., a compound that is structurally and/or functionallysimilar to a natural substrate of a feline FcεRα protein of the presentinvention, particularly to the region of the substrate that binds to afeline FcεRα protein, but that inhibits IgE binding upon interactingwith the IgE binding site).

Feline FcεRα molecules, as well as other inhibitors and therapeuticcompounds, can be used directly as compounds in compositions of thepresent invention to treat animals as long as such compounds are notharmful to the animals being treated.

The present invention also includes a therapeutic composition comprisingone or more therapeutic compounds of the present invention. Examples ofsuch therapeutic compounds are disclosed herein.

In one embodiment, a therapeutic composition of the present inventioncan be used to reduce a Fc epsilon receptor-mediated biological responsein an animal by administering such a composition to an animal.Preferably, an animal is treated by administering to the animal atherapeutic composition of the present invention in such a manner that atherapeutic compound (e.g., an inhibitor of a feline FcεRα protein, ananti-feline FcεRα antibody, an inhibitor of IgE, or nucleic acidmolecules encoding feline FcεRα proteins) binds to an IgE or a Fcepsilon receptor in the animal. Such administration could be by avariety of routes known to those skilled in the art including, but notlimited to, subcutaneous, intradermal, intravenous, intranasal, oral,transdermal, intramuscular routes and other parenteral routes.

Compositions of the present invention can be administered to any animalhaving a Fc epsilon receptor or an IgE that binds to a therapeuticcompound of the present invention or to a protein expressed by a nucleicacid molecule contained in a therapeutic composition. Preferred animalsto treat include mammals and birds, with cats, dogs, horses, humans andother pets, work and/or economic food animals. Particularly preferredanimals to protect are cats and dogs.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, --or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservatives, etc., towhich sterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and compounds that induce the production of cytokines andchemokines (e.g., granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophagecolony stimulating factor (M-CSF), colony stimulating factor (CSF),erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6),interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10),interleukin 12 (IL-12), interferon gamma, interferon gamma inducingfactor I (IGIF), transforming growth factor beta, RANTES (regulated uponactivation, normal T cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of an animal at a constant rate sufficient to attain therapeuticdose levels of the composition to reduce Fc epsilon receptor-mediatedbiological responses in the animal. As used herein, a Fc epsilonreceptor-mediated biological response refers to cellular responses thatoccur when Fc epsilon receptor is complexed with IgE. For example, a Fcepsilon receptor-mediated biological response includes release ofbiological mediators, such as histamine, prostaglandins and/orproteases, that can trigger clinical symptoms of allergy. Thetherapeutic composition is preferably released over a period of timeranging from about 1 to about 12 months. A preferred controlled releaseformulation of the present invention is capable of effecting a treatmentpreferably for at least about 1 month, more preferably for at leastabout 3 months, even more preferably for at least about 6 months, evenmore preferably for at least about 9 months, and even more preferablyfor at least about 12 months.

Acceptable protocols to administer therapeutic compositions of thepresent invention in an effective manner include individual dose size,number of doses, frequency of dose administration, and mode ofadministration. Determination of such protocols can be accomplished bythose skilled in the art. A suitable single dose is a dose that iscapable of protecting (i.e., preventing or treating) an animal fromdisease when administered one or more times over a suitable time period.The need for additional administrations of a therapeutic composition canbe determined by one of skill in the art in accordance with the givencondition of a patient. For example, to regulate an antigen-specific Fcepsilon receptor-mediated response, a therapeutic composition may beadministered more frequently when an antigen is present in a patient'senvironment in high amounts and less frequently when the antigen ispresent in lower amounts.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a feline FcεRα protein ora feline FcεRα RNA (e.g., antisense RNA, ribozyme, triple helix forms orRNA drug) in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked (i.e., not packaged in a viral coat or cellularmembrane) nucleic acid molecule (e.g., as naked DNA or RNA molecules,such as is taught, for example in Wolff et al., 1990, Science 247,1465-1468) or (b) administering a nucleic acid molecule packaged as arecombinant virus or as a recombinant cell (i.e., the nucleic acidmolecule is delivered by a viral or cellular vehicle).

A naked nucleic acid molecule of the present invention includes anucleic acid molecule of the present invention and preferably includes arecombinant molecule of the present invention that preferably isreplication, or otherwise amplification, competent. A naked nucleic acidof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a bicistronicrecombinant molecule having, for example one or more internal ribosomeentry sites. Preferred naked nucleic acid molecules include at least aportion of a viral genome (i.e., a viral vector). Preferred viralvectors include those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses, and retroviruses, with those based onalphaviruses (such as Sindbis or Semliki virus), species-specificherpesviruses and species-specific poxviruses being particularlypreferred. Any suitable transcription control sequence can be used,including those disclosed as suitable for protein production.Particularly preferred transcription control sequence includecytomegalovirus intermediate early (preferably in conjunction withIntron-A), Rous Sarcoma Virus long terminal repeat, and tissue-specifictranscription control sequences, as well as transcription controlsequences endogenous to viral vectors if viral vectors are used. Theincorporation of "strong" poly(A) sequences are also preferred.

Naked nucleic acid molecules of the present invention can beadministered by a variety of methods. Suitable delivery methods include,for example, intramuscular injection, subcutaneous injection,intradermal injection, intradermal scarification, particle bombardment,oral application, and nasal application, with intramuscular injection,intradermal injection, intradermal scarification and particlebombardment being preferred. A preferred single dose of a naked DNAmolecule ranges from about 1 nanogram (ng) to about 1 milligram (mg),depending on the route of administration and/or method of delivery, ascan be determined by those skilled in the art. Examples ofadministration methods are disclosed, for example, in U.S. Pat. No.5,204,253, by Bruner, et al., issued Apr. 20, 1993, PCT Publication No.WO 95/19799, published Jul. 27, 1995, by McCabe, and PCT Publication No.WO 95/05853, published Mar. 2, 1995, by Carson, et al. Naked DNAmolecules of the present invention can be contained in an aqueousexcipient (e.g., phosphate buffered saline) and/or with a carrier (e.g.,lipid-based vehicles), or it can be bound to microparticles (e.g., goldparticles).

A recombinant virus of the present invention includes a recombinantmolecule of the present invention that is packaged in a viral coat andthat can be expressed in an animal after administration. Preferably, therecombinant molecule is packaging-deficient and/or encodes an attenuatedvirus. A number of recombinant viruses can be used, including, but notlimited to, those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picornaviruses and retroviruses. Preferred recombinantviruses are those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. An example of methods to produce and use alphavirusrecombinant virus is disclosed in PCT Publication No. WO 94/17813, byXiong et al., published Aug. 18, 1994, which is incorporated byreference herein in its entirety.

When administered to an animal, a recombinant virus of the presentinvention infects cells within the recipient animal and directs theproduction of a protein or RNA nucleic acid molecule that is capable ofreducing Fc epsilon receptor-mediated biological responses in theanimal. For example, a recombinant virus comprising a feline FcεRαnucleic acid molecule of the present invention is administered accordingto a protocol that results in the animal producing an amount of proteinor RNA sufficient to reduce Fc epsilon receptor-mediated biologicalresponses. A preferred single dose of a recombinant virus of the presentinvention is from about 1×10⁴ to about 1×10⁷ virus plaque forming units(pfu) per kilogram body weight of the animal. Administration protocolsare similar to those described herein for protein-based compositions,with subcutaneous, intramuscular, intranasal and oral administrationroutes being preferred.

A recombinant cell useful in a therapeutic composition of the presentinvention includes recombinant cells of the present invention thatcomprises at least one feline FcεRα of the present invention. Preferredrecombinant cells for this embodiment include Salmonella, E. coli,Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomycescerevisiae), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK andCRFK recombinant cells. A recombinant cell of the present invention canbe administered in a variety of ways but have the advantage that theycan be administered orally, preferably at doses ranging from about 10⁸to about 10¹² cells per kilogram body weight. Administration protocolsare similar to those described herein for protein compositions.Recombinant cells can comprise whole cells, cells stripped of cell wallsor cell lysates.

One embodiment of the present invention is a method of immunotherapycomprising the steps of: (a) administering to an animal an effectiveamount of a therapeutic composition selected from the group consistingof an inhibitor of a feline FcεRα and a feline FcεRα protein (includinghomologs), wherein said feline FcεRα is capable of binding to IgE.Suitable therapeutic compositions and methods of administration methodsare disclosed herein. According to the present invention, a therapeuticcomposition and method of the present invention can be used to preventor alleviate symptoms associated with Fc epsilon receptor-mediatedbiological responses.

The efficacy of a therapeutic composition of the present invention toeffect Fc epsilon receptor-mediated biological responses can be testedusing standard methods for detecting Fc receptor-mediated immunityincluding, but not limited to, immediate hypersensitivity, delayedhypersensitivity, antibody-dependent cellular cytotoxicity (ADCC),immune complex activity, mitogenic activity, histamine release assaysand other methods such as those described in Janeway et al., ibid.

An inhibitor of feline FcεRα activity can be identified using felineFcεRα proteins of the present invention by determining the ability of aninhibitor to prevent or disrupt complex formation between a feline FcεRαprotein and IgE. One embodiment of the present invention is a method toidentify a compound capable of inhibiting feline FcεRα. activity. Such amethod includes the steps of (a) contacting (e.g., combining, mixing) anisolated feline FcεRα protein with a putative inhibitory compound underconditions in which, in the absence of the compound, the feline FcεRαprotein has IgE binding activity, and (b) determining if the putativeinhibitory compound inhibits the IgE binding activity. Putativeinhibitory compounds to screen include small organic molecules,antibodies (including mimetopes thereof) and substrate analogs. Methodsto determine IgE binding activity are known to those skilled in the art.

The present invention also includes a test kit to identify a compoundcapable of inhibiting feline FcεRα activity. Such a test kit includes:an isolated feline FcεRα protein having IgE binding activity or acomplex of feline FcεRα protein and IgE; and a means for determining theextent of inhibition of IgE binding activity in the presence of (i.e.,effected by) a putative inhibitory compound. Such compounds are alsoscreened to identify those that are substantially not toxic in animals.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the Examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques consideredto be known to those skilled in the art. Disclosure of such techniquescan be found, for example, in Sambrook et al., ibid., and relatedreferences.

Example 1

This example describes the isolation, by DNA hybridization, of a nucleicacid molecule encoding a feline Fc epsilon receptor alpha chain (FcεRα)protein from Felis domesticus.

A feline FcεRα nucleic acid molecule was isolated from a feline (Felisdomesticus) mastocytoma cDNA library by hybridizing the library with amixture of ³² P-labeled cDNA molecules encoding human and canine Fcepsilon receptor alpha chains, respectively. A feline mastocytoma cDNAlibrary was prepared as follows. Total RNA was extracted fromapproximately 1.5 grams of tissue from a freshly harvested felinemastocytoma, using an acid-guanidinium-phenol-chloroform method similarto that described by Chomzynski et al., 1987, Anal. Biochem.162,156-159. Poly A⁺ selected RNA was separated from the total RNApreparation by oligo-dT cellulose chromatography using the mRNAPurification Kit (available from Pharmacia Biotech, Newark, N.J.;according to the method recommended by the manufacturer). A felinemastocytoma cDNA library was constructed in lambda-Uni-ZAP™ XR vector(available from Stratagene Cloning Systems, La Jolla, Calif.), usingStratagene's ZAP-cDNA Synthesis Kit protocol. Approximately 5 μg offeline mastocytoma Poly A⁺ RNA was used to produce the felinemastocytoma cDNA library. Using a modification of the protocol describedin the cDNA Synthesis Kit, the feline mastocytoma cDNA library wasscreened, using duplicate plaque lifts, with a mixture of ³² P-labeledcDNAs encoding the human Fc epsilon receptor alpha chain (Kochan et al.,Nucleic Acids Res., 16:3584, 1988) and the canine Fc epsilon receptoralpha chain (Hayashi et al., GenBank accession number D16413, 1993),repsectively. A plaque purified clone identified using the abovescreening method was converted into a double stranded recombinantmolecule, herein denoted as nfelFc.sub.ε Rα₁₀₆₉, using ExAssist™ helperphage and SOLR™ E. coli according to the in vivo excision protocoldescribed in the ZAP-cDNA Synthesis Kit (available from Stratagene).Double-stranded plasmid DNA was prepared using an alkaline lysisprotocol, such as that described in Sambrook et al., ibid.

Example 2

This example describes the sequencing of plasmid DNA containingnfelFc.sub.ε Rα₁₀₆₉. Plasmid DNA containing nfelFc.sub.ε Rα₁₀₆₉ wassequenced by the Sanger dideoxy chain termination method, using thePRISM™ Ready Dye Terminator Cycle Sequencing Kit with Ampli Taq DNAPolymerase, FS (available from the Perkin-Elmer Corporation, Norwalk,Conn.). PCR extensions were done in the GeneAmp™ PCR System 9600(available from Perkin-Elmer). Excess dye terminators were removed fromextension products using the Centriflex™ Gel Filtration Cartridge(available from Advanced Genetics Technologies Corporation,Gaithersburg, Md.) following their standard protocol. Samples wereresuspended according to ABI protocols and were run on a Perkin-ElmerABI PRISM™ 377 Automated DNA Sequencer. DNA sequence analysis, includingthe compilation of sequences and the determination of open readingframes, were performed using the MacVector™ program (available from theEastman Kodak Company, New Haven, Conn.), or the DNAsis™ program(available from Hitachi Software, San Bruno, Calif.). Protein sequenceanalysis, including the determination of molecular weight andisoelectric point (pI) was performed using the MacVector™ program. Anabout 1069 nucleotide consensus sequence of the entire nfelFc.sub.εRα₁₀₆₉ DNA was determined; the sequences of the two complementarystrands are presented as SEQ ID NO:1 (the coding strand) and SEQ ID NO:3(the complementary strand). The nfelFc.sub.ε Rα₁₀₆₉ sequence contains afull length coding region. The apparent initiation (start) codon spanfrom about nucleotide 65 to about nucleotide 67 and the apparenttermination (stop) codon spans from about nucleotide 854 to aboutnucleotide 856, respectively, of SEQ ID NO:1. A putative polyadenylationsignal (5' AATAAA 3') is located in a region spanning from aboutnucleotide 1032 to about nucleotide 1037 of SEQ ID NO:1. Translation ofSEQ ID NO:1 yields a protein of about 263 amino acids, denotedPfelFc.sub.ε Rα₂₆₃, the amino acid sequence of which is presented in SEQID NO:2. The nucleic acid molecule consisting of the coding regionencoding PfelFc.sub.ε Rα₂₆₃ is referred to herein as nfelFc.sub.ε Rα₇₈₉,the nucleic acid sequence of which is represented in SEQ ID NO:4 (thecoding strand) and SEQ ID NO:5 (the complementary strand). The aminoacid sequence of PfelFc.sub.ε Rα₂₆₃ (i.e., SEQ ID NO:2) predicts thatPfelFc.sub.ε Rα₂₆₃ has an estimated molecular weight of about 30.2 kDand an estimated pI of about 9.51. Analysis of SEQ ID NO:2 suggests thepresence of a signal peptide encoded by a stretch of amino acidsspanning from about amino acid 1 through about amino acid 25. Theproposed mature protein, denoted herein as PfelFc.sub.ε Rα₂₃₈, containsabout 238 amino acids which is represented herein as SEQ ID NO:7. Theamino acid sequence of PfelFc.sub.ε Rα₂₃₈ (i.e., SEQ ID NO:7) predictsthat PfelFc.sub.ε Rα₂₃₈ has an estimated molecular weight of about 27.5kD, an estimated pI of about 9.59 and five predicted asparagine-linkedglycosylation sites extending from about amino acids 30-32, 36-38,43-45, 136-138 and 141-143 respectively.

Homology searches of the non-redundant protein and nucleotide sequencedatabases were performed through the National Center for BiotechnologyInformation using the BLAST network. The protein database includesSwissProt+PIR+SPUpdate+Genpept+GPUpdate. The nucleotide databaseincludes GenBank+EMBL+DDBJ+PDB. The highest scoring match of thehomology search at the amino acid level was GenBank accession numberJ03605: Homo Sapiens, which was about 54% identical with SEQ ID NO:2. Atthe nucleotide level, the search was performed using SEQ ID NO:1, whichwas most similar to GenBank accession number D16413, canine (i.e., dog)mRNA for immunoglobulin E receptor alpha chain, there being about 77%identity between feline and canine sequences.

Example 3

This Example demonstrates the production of secreted feline FcεRα chainprotein in eukaryotic cells.

To produce a secreted form of the extracellular domain of the felineFcεRα chain, the hydrophobic transmembrane domain and the cytoplasmictail of the feline FcεRα chain encoded by nfelFc.sub.ε Rα₁₀₆₉ wereremoved as follows. A feline FcεRα chain extracellular domain nucleicacid molecule-containing a fragment of about 597 nucleotides was PCRamplified from nfelFc.sub.ε Rα₁₀₆₉ using a sense primer fellgEr FWDhaving the nucleic acid sequence 5' CGC GAA TTC TATAAA TAT GCC GGT TTTCCT GGG AGG CCCTGC 3' (SEQ ID NO:9; EcoRI site shown in bold) and anantisense primer fellgEr REV having the nucleic acid sequence 5' GCG AGATCT TTA GGA ATC TTT TCT CAC AAC GAT GTT GAG G 3' (SEQ ID NO:10; BglIIsite shown in bold). The resulting PCR product (referred to asBv-nfelFc.sub.ε Rα₅₉₇) was digested with EcoRI and BglII and subclonedinto unique with EcoRI and BglII sites of pVL 1392 baculovirus shuttleplasmid (available from Pharmingen, San Diego, Calif.) to produce therecombinant molecule referred to herein as pVL-nfelFc.sub.ε Rα₅₉₇.Nucleic acid molecule Bv-nfelFc.sub.ε Rα₅₉₇ contained an about 597nucleotide fragment encoding the extracellular domain of the felineFcεRα chain, extending from about nucleotide 65 through about 661 of SEQID NO:1, denoted herein as nucleic acid molecule nfelFc.sub.ε Rα₅₉₇, thecoding strand of which has a nucleic acid sequence denoted SEQ ID NO:11.Translation of SEQ ID NO:11 indicates that nucleic acid moleculenfelFc.sub.ε Rα₅₉₇ encodes a FcεRα protein of about 199 amino acids,referred to herein as PfelFc.sub.ε Rα₁₉₉, having amino acid sequence SEQID NO:12. Nucleic acid molecule nfelFc.sub.ε Rα₅₉₇ encodes a secretableform of the feline FcεRα chain. The processed protein product encoded bynfelFc.sub.ε Rα₅₉₇ is about 174 amino acids and does not possess aleader sequence or a transmembrane domain. Such processed protein isdenoted herein as PfelFc.sub.ε Rα₁₇₄ having amino acid sequence SEQ IDNO:13. The coding region for PfelFc.sub.ε Rα₁₇₄ is denoted nfelFc.sub.εRα₅₂₂, the coding strand of which has a nucleic acid sequence denotedSEQ ID NO:14. The complement of SEQ ID NO:14 is represented herein bySEQ ID NO:15.

The resultant recombinant molecule, pVL-nfelFc.sub.ε Rα₅₉₇, was verifiedfor proper insert orientation by restriction mapping. Such a recombinantmolecule was co-transfected with a linear Baculogold baculovirus DNA(available from Pharmingen) into S. frugiperda Sf9 cells (available fromInVitrogen) to form a recombinant cell denoted S.frugiperda:pVL-nfelFc.sub.ε Rα₅₉₇. S. frugiperda:pVL-nfelFc.sub.ε Rα₅₉₇is cultured using techniques known to those skilled in the art toproduce a feline FcεRα protein PfelFc.sub.ε Rα₁₉₉.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  16    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1069 nu - #cleotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (ix) FEATURE:              (A) NAME/KEY:  CDS              (B) LOCATION:  65..856    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:    - TTTAAGTCTA TTTTAAGGCG TTAGGTCTCT CCCGTCGGGT CGGCATTTGG GA - #GCCAGGGA      60    - GGCG ATG CCG GTT TTC CTG GGA GGC CCT GCT C - #TG CTG TGG ACA GCA CTG     109         Met Pro Val Phe Leu Gly Gly Pro - # Ala Leu Leu Trp Thr Ala Leu    #    15    - CTG CTC CTC CTC TAT CCA GAT GGC ATG TCA GC - #A GGC ACC CGG GAA CCT     157    Leu Leu Leu Leu Tyr Pro Asp Gly Met Ser Al - #a Gly Thr Arg Glu Pro    #                 30    - ACA GTG TCC TTG AAT CCA CCG TGG ACT ACC AT - #A TTG AAA GAA GAC AGT     205    Thr Val Ser Leu Asn Pro Pro Trp Thr Thr Il - #e Leu Lys Glu Asp Ser    #             45    - GTG ACT CTT ACA TGT AAA GAG AAC AAT TCT CT - #T GAA CTC AAC TCT ACT     253    Val Thr Leu Thr Cys Lys Glu Asn Asn Ser Le - #u Glu Leu Asn Ser Thr    #         60    - GTG TGG TTC CAC AAC AAG ACC AAG TTG GGA GT - #G ACA ACT TTA ACT TTG     301    Val Trp Phe His Asn Lys Thr Lys Leu Gly Va - #l Thr Thr Leu Thr Leu    #     75    - GAC ATC GTG AAA GCC CAA ATC CGC GAT AGT GG - #G GAA TAC ACG TGT CAG     349    Asp Ile Val Lys Ala Gln Ile Arg Asp Ser Gl - #y Glu Tyr Thr Cys Gln    # 95    - AAC AAA GGA TCC ATG CTG AGT AAA CCT GTG TC - #C TTA AAA GTC TTC CGT     397    Asn Lys Gly Ser Met Leu Ser Lys Pro Val Se - #r Leu Lys Val Phe Arg    #               110    - GAG TGG CTG CTC CTT CAG GCC TCT ACT GAG GT - #G GTG CTG GAG GGT GAG     445    Glu Trp Leu Leu Leu Gln Ala Ser Thr Glu Va - #l Val Leu Glu Gly Glu    #           125    - TCC TTC CTC ATC AGG TGC CAC AGT TGG AGG AA - #T TTG AAT GTC AAA AAA     493    Ser Phe Leu Ile Arg Cys His Ser Trp Arg As - #n Leu Asn Val Lys Lys    #       140    - GTG ACC TAC TAC AGG AAT GGC AAG TTC CTC CA - #G TTC TGG TAC GAC AAC     541    Val Thr Tyr Tyr Arg Asn Gly Lys Phe Leu Gl - #n Phe Trp Tyr Asp Asn    #   155    - TAC AAC ATC ACC ATT AAC AAT GCC ACA GAA AC - #A GAC AGC GGC ACC TAC     589    Tyr Asn Ile Thr Ile Asn Asn Ala Thr Glu Th - #r Asp Ser Gly Thr Tyr    160                 1 - #65                 1 - #70                 1 -    #75    - TAC TGC ACG GGC TGG ATT TCG AGG CAA AAT CA - #C ATC TCT AAC TTC CTC     637    Tyr Cys Thr Gly Trp Ile Ser Arg Gln Asn Hi - #s Ile Ser Asn Phe Leu    #               190    - AAC ATC GTT GTG AGA AAA GAT TCC CCT CCG GA - #G CAC CAA AGC AAA TAC     685    Asn Ile Val Val Arg Lys Asp Ser Pro Pro Gl - #u His Gln Ser Lys Tyr    #           205    - TAC TGG CTA CAA TTT GTG ATC CCA TCG TTG GT - #G GTG CTT CTG TTT GCT     733    Tyr Trp Leu Gln Phe Val Ile Pro Ser Leu Va - #l Val Leu Leu Phe Ala    #       220    - GCG GAC ACG GGG CTG TTT ATC TCG ACC CAG CA - #G CAG CTG ACC CTG CTC     781    Ala Asp Thr Gly Leu Phe Ile Ser Thr Gln Gl - #n Gln Leu Thr Leu Leu    #   235    - TTG AAG ATT AAG ACG ACC AGG AGG AGC AGG AA - #C CTT ATG GAC CCA CGT     829    Leu Lys Ile Lys Thr Thr Arg Arg Ser Arg As - #n Leu Met Asp Pro Arg    240                 2 - #45                 2 - #50                 2 -    #55    - CCC AAG CCA GAC CCC AAA AAG AAC TGA TGTTGCTGC - #T TGGGAAACAT     876    Pro Lys Pro Asp Pro Lys Lys Asn                    260    - TTGCAACGGC AACCTCTTTC TGGCATCAGC GATTGCTTCT CCGTGGTCAA AC - #ACAGCTCG     936    - CAACGCACAC AGGAGCGTCT GGACGCAAGG CTTTAACAGA CCTGCTTCAT TA - #AGCCAGCT     996    - GAAACTGGTT ACATGGCATG TAACAACAAG GGCTCAATAA ACATCACTTA AA - #CAAAAAAA    1056    #    1069    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  263 ami - #no acids              (B) TYPE:  amino aci - #d              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  protein    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:    - Met Pro Val Phe Leu Gly Gly Pro Ala Leu Le - #u Trp Thr Ala Leu Leu    #                 15    - Leu Leu Leu Tyr Pro Asp Gly Met Ser Ala Gl - #y Thr Arg Glu Pro Thr    #             30    - Val Ser Leu Asn Pro Pro Trp Thr Thr Ile Le - #u Lys Glu Asp Ser Val    #         45    - Thr Leu Thr Cys Lys Glu Asn Asn Ser Leu Gl - #u Leu Asn Ser Thr Val    #     60    - Trp Phe His Asn Lys Thr Lys Leu Gly Val Th - #r Thr Leu Thr Leu Asp    # 80    - Ile Val Lys Ala Gln Ile Arg Asp Ser Gly Gl - #u Tyr Thr Cys Gln Asn    #                 95    - Lys Gly Ser Met Leu Ser Lys Pro Val Ser Le - #u Lys Val Phe Arg Glu    #           110    - Trp Leu Leu Leu Gln Ala Ser Thr Glu Val Va - #l Leu Glu Gly Glu Ser    #       125    - Phe Leu Ile Arg Cys His Ser Trp Arg Asn Le - #u Asn Val Lys Lys Val    #   140    - Thr Tyr Tyr Arg Asn Gly Lys Phe Leu Gln Ph - #e Trp Tyr Asp Asn Tyr    145                 1 - #50                 1 - #55                 1 -    #60    - Asn Ile Thr Ile Asn Asn Ala Thr Glu Thr As - #p Ser Gly Thr Tyr Tyr    #               175    - Cys Thr Gly Trp Ile Ser Arg Gln Asn His Il - #e Ser Asn Phe Leu Asn    #           190    - Ile Val Val Arg Lys Asp Ser Pro Pro Glu Hi - #s Gln Ser Lys Tyr Tyr    #       205    - Trp Leu Gln Phe Val Ile Pro Ser Leu Val Va - #l Leu Leu Phe Ala Ala    #   220    - Asp Thr Gly Leu Phe Ile Ser Thr Gln Gln Gl - #n Leu Thr Leu Leu Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Lys Ile Lys Thr Thr Arg Arg Ser Arg Asn Le - #u Met Asp Pro Arg Pro    #               255    - Lys Pro Asp Pro Lys Lys Asn                260    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1069 nu - #cleotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:    - TTTTTTTTTT TTTTTTTTTT GTTTAAGTGA TGTTTATTGA GCCCTTGTTG TT - #ACATGCCA      60    - TGTAACCAGT TTCAGCTGGC TTAATGAAGC AGGTCTGTTA AAGCCTTGCG TC - #CAGACGCT     120    - CCTGTGTGCG TTGCGAGCTG TGTTTGACCA CGGAGAAGCA ATCGCTGATG CC - #AGAAAGAG     180    - GTTGCCGTTG CAAATGTTTC CCAAGCAGCA ACATCAGTTC TTTTTGGGGT CT - #GGCTTGGG     240    - ACGTGGGTCC ATAAGGTTCC TGCTCCTCCT GGTCGTCTTA ATCTTCAAGA GC - #AGGGTCAG     300    - CTGCTGCTGG GTCGAGATAA ACAGCCCCGT GTCCGCAGCA AACAGAAGCA CC - #ACCAACGA     360    - TGGGATCACA AATTGTAGCC AGTAGTATTT GCTTTGGTGC TCCGGAGGGG AA - #TCTTTTCT     420    - CACAACGATG TTGAGGAAGT TAGAGATGTG ATTTTGCCTC GAAATCCAGC CC - #GTGCAGTA     480    - GTAGGTGCCG CTGTCTGTTT CTGTGGCATT GTTAATGGTG ATGTTGTAGT TG - #TCGTACCA     540    - GAACTGGAGG AACTTGCCAT TCCTGTAGTA GGTCACTTTT TTGACATTCA AA - #TTCCTCCA     600    - ACTGTGGCAC CTGATGAGGA AGGACTCACC CTCCAGCACC ACCTCAGTAG AG - #GCCTGAAG     660    - GAGCAGCCAC TCACGGAAGA CTTTTAAGGA CACAGGTTTA CTCAGCATGG AT - #CCTTTGTT     720    - CTGACACGTG TATTCCCCAC TATCGCGGAT TTGGGCTTTC ACGATGTCCA AA - #GTTAAAGT     780    - TGTCACTCCC AACTTGGTCT TGTTGTGGAA CCACACAGTA GAGTTGAGTT CA - #AGAGAATT     840    - GTTCTCTTTA CATGTAAGAG TCACACTGTC TTCTTTCAAT ATGGTAGTCC AC - #GGTGGATT     900    - CAAGGACACT GTAGGTTCCC GGGTGCCTGC TGACATGCCA TCTGGATAGA GG - #AGGAGCAG     960    - CAGTGCTGTC CACAGCAGAG CAGGGCCTCC CAGGAAAACC GGCATCGCCT CC - #CTGGCTCC    1020    #             1069CGGGA GAGACCTAAC GCCTTAAAAT AGACTTAAA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  789 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (ix) FEATURE:              (A) NAME/KEY:  CDS              (B) LOCATION:  1..789    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:    - ATG CCG GTT TTC CTG GGA GGC CCT GCT CTG CT - #G TGG ACA GCA CTG CTG      48    Met Pro Val Phe Leu Gly Gly Pro Ala Leu Le - #u Trp Thr Ala Leu Leu    #   15    - CTC CTC CTC TAT CCA GAT GGC ATG TCA GCA GG - #C ACC CGG GAA CCT ACA      96    Leu Leu Leu Tyr Pro Asp Gly Met Ser Ala Gl - #y Thr Arg Glu Pro Thr    #                 30    - GTG TCC TTG AAT CCA CCG TGG ACT ACC ATA TT - #G AAA GAA GAC AGT GTG     144    Val Ser Leu Asn Pro Pro Trp Thr Thr Ile Le - #u Lys Glu Asp Ser Val    #             45    - ACT CTT ACA TGT AAA GAG AAC AAT TCT CTT GA - #A CTC AAC TCT ACT GTG     192    Thr Leu Thr Cys Lys Glu Asn Asn Ser Leu Gl - #u Leu Asn Ser Thr Val    #         60    - TGG TTC CAC AAC AAG ACC AAG TTG GGA GTG AC - #A ACT TTA ACT TTG GAC     240    Trp Phe His Asn Lys Thr Lys Leu Gly Val Th - #r Thr Leu Thr Leu Asp    #     75    - ATC GTG AAA GCC CAA ATC CGC GAT AGT GGG GA - #A TAC ACG TGT CAG AAC     288    Ile Val Lys Ala Gln Ile Arg Asp Ser Gly Gl - #u Tyr Thr Cys Gln Asn    # 95    - AAA GGA TCC ATG CTG AGT AAA CCT GTG TCC TT - #A AAA GTC TTC CGT GAG     336    Lys Gly Ser Met Leu Ser Lys Pro Val Ser Le - #u Lys Val Phe Arg Glu    #               110    - TGG CTG CTC CTT CAG GCC TCT ACT GAG GTG GT - #G CTG GAG GGT GAG TCC     384    Trp Leu Leu Leu Gln Ala Ser Thr Glu Val Va - #l Leu Glu Gly Glu Ser    #           125    - TTC CTC ATC AGG TGC CAC AGT TGG AGG AAT TT - #G AAT GTC AAA AAA GTG     432    Phe Leu Ile Arg Cys His Ser Trp Arg Asn Le - #u Asn Val Lys Lys Val    #       140    - ACC TAC TAC AGG AAT GGC AAG TTC CTC CAG TT - #C TGG TAC GAC AAC TAC     480    Thr Tyr Tyr Arg Asn Gly Lys Phe Leu Gln Ph - #e Trp Tyr Asp Asn Tyr    #   155    - AAC ATC ACC ATT AAC AAT GCC ACA GAA ACA GA - #C AGC GGC ACC TAC TAC     528    Asn Ile Thr Ile Asn Asn Ala Thr Glu Thr As - #p Ser Gly Thr Tyr Tyr    160                 1 - #65                 1 - #70                 1 -    #75    - TGC ACG GGC TGG ATT TCG AGG CAA AAT CAC AT - #C TCT AAC TTC CTC AAC     576    Cys Thr Gly Trp Ile Ser Arg Gln Asn His Il - #e Ser Asn Phe Leu Asn    #               190    - ATC GTT GTG AGA AAA GAT TCC CCT CCG GAG CA - #C CAA AGC AAA TAC TAC     624    Ile Val Val Arg Lys Asp Ser Pro Pro Glu Hi - #s Gln Ser Lys Tyr Tyr    #           205    - TGG CTA CAA TTT GTG ATC CCA TCG TTG GTG GT - #G CTT CTG TTT GCT GCG     672    Trp Leu Gln Phe Val Ile Pro Ser Leu Val Va - #l Leu Leu Phe Ala Ala    #       220    - GAC ACG GGG CTG TTT ATC TCG ACC CAG CAG CA - #G CTG ACC CTG CTC TTG     720    Asp Thr Gly Leu Phe Ile Ser Thr Gln Gln Gl - #n Leu Thr Leu Leu Leu    #   235    - AAG ATT AAG ACG ACC AGG AGG AGC AGG AAC CT - #T ATG GAC CCA CGT CCC     768    Lys Ile Lys Thr Thr Arg Arg Ser Arg Asn Le - #u Met Asp Pro Arg Pro    240                 2 - #45                 2 - #50                 2 -    #55    #                 789 AAG AAC    Lys Pro Asp Pro Lys Lys Asn                    260    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  789 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:    - GTTCTTTTTG GGGTCTGGCT TGGGACGTGG GTCCATAAGG TTCCTGCTCC TC - #CTGGTCGT      60    - CTTAATCTTC AAGAGCAGGG TCAGCTGCTG CTGGGTCGAG ATAAACAGCC CC - #GTGTCCGC     120    - AGCAAACAGA AGCACCACCA ACGATGGGAT CACAAATTGT AGCCAGTAGT AT - #TTGCTTTG     180    - GTGCTCCGGA GGGGAATCTT TTCTCACAAC GATGTTGAGG AAGTTAGAGA TG - #TGATTTTG     240    - CCTCGAAATC CAGCCCGTGC AGTAGTAGGT GCCGCTGTCT GTTTCTGTGG CA - #TTGTTAAT     300    - GGTGATGTTG TAGTTGTCGT ACCAGAACTG GAGGAACTTG CCATTCCTGT AG - #TAGGTCAC     360    - TTTTTTGACA TTCAAATTCC TCCAACTGTG GCACCTGATG AGGAAGGACT CA - #CCCTCCAG     420    - CACCACCTCA GTAGAGGCCT GAAGGAGCAG CCACTCACGG AAGACTTTTA AG - #GACACAGG     480    - TTTACTCAGC ATGGATCCTT TGTTCTGACA CGTGTATTCC CCACTATCGC GG - #ATTTGGGC     540    - TTTCACGATG TCCAAAGTTA AAGTTGTCAC TCCCAACTTG GTCTTGTTGT GG - #AACCACAC     600    - AGTAGAGTTG AGTTCAAGAG AATTGTTCTC TTTACATGTA AGAGTCACAC TG - #TCTTCTTT     660    - CAATATGGTA GTCCACGGTG GATTCAAGGA CACTGTAGGT TCCCGGGTGC CT - #GCTGACAT     720    - GCCATCTGGA TAGAGGAGGA GCAGCAGTGC TGTCCACAGC AGAGCAGGGC CT - #CCCAGGAA     780    #        789    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  714 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (ix) FEATURE:              (A) NAME/KEY:  CDS              (B) LOCATION:  1..714    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:    - GCA GGC ACC CGG GAA CCT ACA GTG TCC TTG AA - #T CCA CCG TGG ACT ACC      48    Ala Gly Thr Arg Glu Pro Thr Val Ser Leu As - #n Pro Pro Trp Thr Thr    #                 15    - ATA TTG AAA GAA GAC AGT GTG ACT CTT ACA TG - #T AAA GAG AAC AAT TCT      96    Ile Leu Lys Glu Asp Ser Val Thr Leu Thr Cy - #s Lys Glu Asn Asn Ser    #             30    - CTT GAA CTC AAC TCT ACT GTG TGG TTC CAC AA - #C AAG ACC AAG TTG GGA     144    Leu Glu Leu Asn Ser Thr Val Trp Phe His As - #n Lys Thr Lys Leu Gly    #         45    - GTG ACA ACT TTA ACT TTG GAC ATC GTG AAA GC - #C CAA ATC CGC GAT AGT     192    Val Thr Thr Leu Thr Leu Asp Ile Val Lys Al - #a Gln Ile Arg Asp Ser    #     60    - GGG GAA TAC ACG TGT CAG AAC AAA GGA TCC AT - #G CTG AGT AAA CCT GTG     240    Gly Glu Tyr Thr Cys Gln Asn Lys Gly Ser Me - #t Leu Ser Lys Pro Val    # 80    - TCC TTA AAA GTC TTC CGT GAG TGG CTG CTC CT - #T CAG GCC TCT ACT GAG     288    Ser Leu Lys Val Phe Arg Glu Trp Leu Leu Le - #u Gln Ala Ser Thr Glu    #                 95    - GTG GTG CTG GAG GGT GAG TCC TTC CTC ATC AG - #G TGC CAC AGT TGG AGG     336    Val Val Leu Glu Gly Glu Ser Phe Leu Ile Ar - #g Cys His Ser Trp Arg    #           110    - AAT TTG AAT GTC AAA AAA GTG ACC TAC TAC AG - #G AAT GGC AAG TTC CTC     384    Asn Leu Asn Val Lys Lys Val Thr Tyr Tyr Ar - #g Asn Gly Lys Phe Leu    #       125    - CAG TTC TGG TAC GAC AAC TAC AAC ATC ACC AT - #T AAC AAT GCC ACA GAA     432    Gln Phe Trp Tyr Asp Asn Tyr Asn Ile Thr Il - #e Asn Asn Ala Thr Glu    #   140    - ACA GAC AGC GGC ACC TAC TAC TGC ACG GGC TG - #G ATT TCG AGG CAA AAT     480    Thr Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Tr - #p Ile Ser Arg Gln Asn    145                 1 - #50                 1 - #55                 1 -    #60    - CAC ATC TCT AAC TTC CTC AAC ATC GTT GTG AG - #A AAA GAT TCC CCT CCG     528    His Ile Ser Asn Phe Leu Asn Ile Val Val Ar - #g Lys Asp Ser Pro Pro    #               175    - GAG CAC CAA AGC AAA TAC TAC TGG CTA CAA TT - #T GTG ATC CCA TCG TTG     576    Glu His Gln Ser Lys Tyr Tyr Trp Leu Gln Ph - #e Val Ile Pro Ser Leu    #           190    - GTG GTG CTT CTG TTT GCT GCG GAC ACG GGG CT - #G TTT ATC TCG ACC CAG     624    Val Val Leu Leu Phe Ala Ala Asp Thr Gly Le - #u Phe Ile Ser Thr Gln    #       205    - CAG CAG CTG ACC CTG CTC TTG AAG ATT AAG AC - #G ACC AGG AGG AGC AGG     672    Gln Gln Leu Thr Leu Leu Leu Lys Ile Lys Th - #r Thr Arg Arg Ser Arg    #   220    - AAC CTT ATG GAC CCA CGT CCC AAG CCA GAC CC - #C AAA AAG AAC    # 714    Asn Leu Met Asp Pro Arg Pro Lys Pro Asp Pr - #o Lys Lys Asn    225                 2 - #30                 2 - #35    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  238 ami - #no acids              (B) TYPE:  amino aci - #d              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  protein    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:    - Ala Gly Thr Arg Glu Pro Thr Val Ser Leu As - #n Pro Pro Trp Thr Thr    #                 15    - Ile Leu Lys Glu Asp Ser Val Thr Leu Thr Cy - #s Lys Glu Asn Asn Ser    #             30    - Leu Glu Leu Asn Ser Thr Val Trp Phe His As - #n Lys Thr Lys Leu Gly    #         45    - Val Thr Thr Leu Thr Leu Asp Ile Val Lys Al - #a Gln Ile Arg Asp Ser    #     60    - Gly Glu Tyr Thr Cys Gln Asn Lys Gly Ser Me - #t Leu Ser Lys Pro Val    # 80    - Ser Leu Lys Val Phe Arg Glu Trp Leu Leu Le - #u Gln Ala Ser Thr Glu    #                 95    - Val Val Leu Glu Gly Glu Ser Phe Leu Ile Ar - #g Cys His Ser Trp Arg    #           110    - Asn Leu Asn Val Lys Lys Val Thr Tyr Tyr Ar - #g Asn Gly Lys Phe Leu    #       125    - Gln Phe Trp Tyr Asp Asn Tyr Asn Ile Thr Il - #e Asn Asn Ala Thr Glu    #   140    - Thr Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Tr - #p Ile Ser Arg Gln Asn    145                 1 - #50                 1 - #55                 1 -    #60    - His Ile Ser Asn Phe Leu Asn Ile Val Val Ar - #g Lys Asp Ser Pro Pro    #               175    - Glu His Gln Ser Lys Tyr Tyr Trp Leu Gln Ph - #e Val Ile Pro Ser Leu    #           190    - Val Val Leu Leu Phe Ala Ala Asp Thr Gly Le - #u Phe Ile Ser Thr Gln    #       205    - Gln Gln Leu Thr Leu Leu Leu Lys Ile Lys Th - #r Thr Arg Arg Ser Arg    #   220    - Asn Leu Met Asp Pro Arg Pro Lys Pro Asp Pr - #o Lys Lys Asn    225                 2 - #30                 2 - #35    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  714 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:    - GTTCTTTTTG GGGTCTGGCT TGGGACGTGG GTCCATAAGG TTCCTGCTCC TC - #CTGGTCGT      60    - CTTAATCTTC AAGAGCAGGG TCAGCTGCTG CTGGGTCGAG ATAAACAGCC CC - #GTGTCCGC     120    - AGCAAACAGA AGCACCACCA ACGATGGGAT CACAAATTGT AGCCAGTAGT AT - #TTGCTTTG     180    - GTGCTCCGGA GGGGAATCTT TTCTCACAAC GATGTTGAGG AAGTTAGAGA TG - #TGATTTTG     240    - CCTCGAAATC CAGCCCGTGC AGTAGTAGGT GCCGCTGTCT GTTTCTGTGG CA - #TTGTTAAT     300    - GGTGATGTTG TAGTTGTCGT ACCAGAACTG GAGGAACTTG CCATTCCTGT AG - #TAGGTCAC     360    - TTTTTTGACA TTCAAATTCC TCCAACTGTG GCACCTGATG AGGAAGGACT CA - #CCCTCCAG     420    - CACCACCTCA GTAGAGGCCT GAAGGAGCAG CCACTCACGG AAGACTTTTA AG - #GACACAGG     480    - TTTACTCAGC ATGGATCCTT TGTTCTGACA CGTGTATTCC CCACTATCGC GG - #ATTTGGGC     540    - TTTCACGATG TCCAAAGTTA AAGTTGTCAC TCCCAACTTG GTCTTGTTGT GG - #AACCACAC     600    - AGTAGAGTTG AGTTCAAGAG AATTGTTCTC TTTACATGTA AGAGTCACAC TG - #TCTTCTTT     660    - CAATATGGTA GTCCACGGTG GATTCAAGGA CACTGTAGGT TCCCGGGTGC CT - #GC     714    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  42 base - #s              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  primer    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:    #  42              ATGC CGGTTTTCCT GGGAGGCCCT GC    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  40 base - #s              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  primer    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #10:    #    40            TCTT TTCTCACAAC GATGTTGAGG    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  597 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (ix) FEATURE:              (A) NAME/KEY:  CDS              (B) LOCATION:  1..597    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #11:    - ATG CCG GTT TTC CTG GGA GGC CCT GCT CTG CT - #G TGG ACA GCA CTG CTG      48    Met Pro Val Phe Leu Gly Gly Pro Ala Leu Le - #u Trp Thr Ala Leu Leu    #                 15    - CTC CTC CTC TAT CCA GAT GGC ATG TCA GCA GG - #C ACC CGG GAA CCT ACA      96    Leu Leu Leu Tyr Pro Asp Gly Met Ser Ala Gl - #y Thr Arg Glu Pro Thr    #             30    - GTG TCC TTG AAT CCA CCG TGG ACT ACC ATA TT - #G AAA GAA GAC AGT GTG     144    Val Ser Leu Asn Pro Pro Trp Thr Thr Ile Le - #u Lys Glu Asp Ser Val    #         45    - ACT CTT ACA TGT AAA GAG AAC AAT TCT CTT GA - #A CTC AAC TCT ACT GTG     192    Thr Leu Thr Cys Lys Glu Asn Asn Ser Leu Gl - #u Leu Asn Ser Thr Val    #     60    - TGG TTC CAC AAC AAG ACC AAG TTG GGA GTG AC - #A ACT TTA ACT TTG GAC     240    Trp Phe His Asn Lys Thr Lys Leu Gly Val Th - #r Thr Leu Thr Leu Asp    # 80    - ATC GTG AAA GCC CAA ATC CGC GAT AGT GGG GA - #A TAC ACG TGT CAG AAC     288    Ile Val Lys Ala Gln Ile Arg Asp Ser Gly Gl - #u Tyr Thr Cys Gln Asn    #                 95    - AAA GGA TCC ATG CTG AGT AAA CCT GTG TCC TT - #A AAA GTC TTC CGT GAG     336    Lys Gly Ser Met Leu Ser Lys Pro Val Ser Le - #u Lys Val Phe Arg Glu    #           110    - TGG CTG CTC CTT CAG GCC TCT ACT GAG GTG GT - #G CTG GAG GGT GAG TCC     384    Trp Leu Leu Leu Gln Ala Ser Thr Glu Val Va - #l Leu Glu Gly Glu Ser    #       125    - TTC CTC ATC AGG TGC CAC AGT TGG AGG AAT TT - #G AAT GTC AAA AAA GTG     432    Phe Leu Ile Arg Cys His Ser Trp Arg Asn Le - #u Asn Val Lys Lys Val    #   140    - ACC TAC TAC AGG AAT GGC AAG TTC CTC CAG TT - #C TGG TAC GAC AAC TAC     480    Thr Tyr Tyr Arg Asn Gly Lys Phe Leu Gln Ph - #e Trp Tyr Asp Asn Tyr    145                 1 - #50                 1 - #55                 1 -    #60    - AAC ATC ACC ATT AAC AAT GCC ACA GAA ACA GA - #C AGC GGC ACC TAC TAC     528    Asn Ile Thr Ile Asn Asn Ala Thr Glu Thr As - #p Ser Gly Thr Tyr Tyr    #               175    - TGC ACG GGC TGG ATT TCG AGG CAA AAT CAC AT - #C TCT AAC TTC CTC AAC     576    Cys Thr Gly Trp Ile Ser Arg Gln Asn His Il - #e Ser Asn Phe Leu Asn    #           190    #                 597 GAT TCC    Ile Val Val Arg Lys Asp Ser            195    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  199 ami - #no acids              (B) TYPE:  amino aci - #d              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  protein    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #12:    - Met Pro Val Phe Leu Gly Gly Pro Ala Leu Le - #u Trp Thr Ala Leu Leu    #                 15    - Leu Leu Leu Tyr Pro Asp Gly Met Ser Ala Gl - #y Thr Arg Glu Pro Thr    #             30    - Val Ser Leu Asn Pro Pro Trp Thr Thr Ile Le - #u Lys Glu Asp Ser Val    #         45    - Thr Leu Thr Cys Lys Glu Asn Asn Ser Leu Gl - #u Leu Asn Ser Thr Val    #     60    - Trp Phe His Asn Lys Thr Lys Leu Gly Val Th - #r Thr Leu Thr Leu Asp    # 80    - Ile Val Lys Ala Gln Ile Arg Asp Ser Gly Gl - #u Tyr Thr Cys Gln Asn    #                 95    - Lys Gly Ser Met Leu Ser Lys Pro Val Ser Le - #u Lys Val Phe Arg Glu    #           110    - Trp Leu Leu Leu Gln Ala Ser Thr Glu Val Va - #l Leu Glu Gly Glu Ser    #       125    - Phe Leu Ile Arg Cys His Ser Trp Arg Asn Le - #u Asn Val Lys Lys Val    #   140    - Thr Tyr Tyr Arg Asn Gly Lys Phe Leu Gln Ph - #e Trp Tyr Asp Asn Tyr    145                 1 - #50                 1 - #55                 1 -    #60    - Asn Ile Thr Ile Asn Asn Ala Thr Glu Thr As - #p Ser Gly Thr Tyr Tyr    #               175    - Cys Thr Gly Trp Ile Ser Arg Gln Asn His Il - #e Ser Asn Phe Leu Asn    #           190    - Ile Val Val Arg Lys Asp Ser            195    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  174 ami - #no acids              (B) TYPE:  amino aci - #d              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  protein    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #13:    - Ala Gly Thr Arg Glu Pro Thr Val Ser Leu As - #n Pro Pro Trp Thr Thr    #                 15    - Ile Leu Lys Glu Asp Ser Val Thr Leu Thr Cy - #s Lys Glu Asn Asn Ser    #             30    - Leu Glu Leu Asn Ser Thr Val Trp Phe His As - #n Lys Thr Lys Leu Gly    #         45    - Val Thr Thr Leu Thr Leu Asp Ile Val Lys Al - #a Gln Ile Arg Asp Ser    #     60    - Gly Glu Tyr Thr Cys Gln Asn Lys Gly Ser Me - #t Leu Ser Lys Pro Val    # 80    - Ser Leu Lys Val Phe Arg Glu Trp Leu Leu Le - #u Gln Ala Ser Thr Glu    #                 95    - Val Val Leu Glu Gly Glu Ser Phe Leu Ile Ar - #g Cys His Ser Trp Arg    #           110    - Asn Leu Asn Val Lys Lys Val Thr Tyr Tyr Ar - #g Asn Gly Lys Phe Leu    #       125    - Gln Phe Trp Tyr Asp Asn Tyr Asn Ile Thr Il - #e Asn Asn Ala Thr Glu    #   140    - Thr Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Tr - #p Ile Ser Arg Gln Asn    145                 1 - #50                 1 - #55                 1 -    #60    - His Ile Ser Asn Phe Leu Asn Ile Val Val Ar - #g Lys Asp Ser    #               170    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  522 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (ix) FEATURE:              (A) NAME/KEY:  CDS              (B) LOCATION:  1..522    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #14:    - GCA GGC ACC CGG GAA CCT ACA GTG TCC TTG AA - #T CCA CCG TGG ACT ACC      48    Ala Gly Thr Arg Glu Pro Thr Val Ser Leu As - #n Pro Pro Trp Thr Thr    #                 15    - ATA TTG AAA GAA GAC AGT GTG ACT CTT ACA TG - #T AAA GAG AAC AAT TCT      96    Ile Leu Lys Glu Asp Ser Val Thr Leu Thr Cy - #s Lys Glu Asn Asn Ser    #             30    - CTT GAA CTC AAC TCT ACT GTG TGG TTC CAC AA - #C AAG ACC AAG TTG GGA     144    Leu Glu Leu Asn Ser Thr Val Trp Phe His As - #n Lys Thr Lys Leu Gly    #         45    - GTG ACA ACT TTA ACT TTG GAC ATC GTG AAA GC - #C CAA ATC CGC GAT AGT     192    Val Thr Thr Leu Thr Leu Asp Ile Val Lys Al - #a Gln Ile Arg Asp Ser    #     60    - GGG GAA TAC ACG TGT CAG AAC AAA GGA TCC AT - #G CTG AGT AAA CCT GTG     240    Gly Glu Tyr Thr Cys Gln Asn Lys Gly Ser Me - #t Leu Ser Lys Pro Val    # 80    - TCC TTA AAA GTC TTC CGT GAG TGG CTG CTC CT - #T CAG GCC TCT ACT GAG     288    Ser Leu Lys Val Phe Arg Glu Trp Leu Leu Le - #u Gln Ala Ser Thr Glu    #                 95    - GTG GTG CTG GAG GGT GAG TCC TTC CTC ATC AG - #G TGC CAC AGT TGG AGG     336    Val Val Leu Glu Gly Glu Ser Phe Leu Ile Ar - #g Cys His Ser Trp Arg    #           110    - AAT TTG AAT GTC AAA AAA GTG ACC TAC TAC AG - #G AAT GGC AAG TTC CTC     384    Asn Leu Asn Val Lys Lys Val Thr Tyr Tyr Ar - #g Asn Gly Lys Phe Leu    #       125    - CAG TTC TGG TAC GAC AAC TAC AAC ATC ACC AT - #T AAC AAT GCC ACA GAA     432    Gln Phe Trp Tyr Asp Asn Tyr Asn Ile Thr Il - #e Asn Asn Ala Thr Glu    #   140    - ACA GAC AGC GGC ACC TAC TAC TGC ACG GGC TG - #G ATT TCG AGG CAA AAT     480    Thr Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Tr - #p Ile Ser Arg Gln Asn    145                 1 - #50                 1 - #55                 1 -    #60    - CAC ATC TCT AAC TTC CTC AAC ATC GTT GTG AG - #A AAA GAT TCC    # 522    His Ile Ser Asn Phe Leu Asn Ile Val Val Ar - #g Lys Asp Ser    #               170    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  522 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #15:    - GGAATCTTTT CTCACAACGA TGTTGAGGAA GTTAGAGATG TGATTTTGCC TC - #GAAATCCA      60    - GCCCGTGCAG TAGTAGGTGC CGCTGTCTGT TTCTGTGGCA TTGTTAATGG TG - #ATGTTGTA     120    - GTTGTCGTAC CAGAACTGGA GGAACTTGCC ATTCCTGTAG TAGGTCACTT TT - #TTGACATT     180    - CAAATTCCTC CAACTGTGGC ACCTGATGAG GAAGGACTCA CCCTCCAGCA CC - #ACCTCAGT     240    - AGAGGCCTGA AGGAGCAGCC ACTCACGGAA GACTTTTAAG GACACAGGTT TA - #CTCAGCAT     300    - GGATCCTTTG TTCTGACACG TGTATTCCCC ACTATCGCGG ATTTGGGCTT TC - #ACGATGTC     360    - CAAAGTTAAA GTTGTCACTC CCAACTTGGT CTTGTTGTGG AACCACACAG TA - #GAGTTGAG     420    - TTCAAGAGAA TTGTTCTCTT TACATGTAAG AGTCACACTG TCTTCTTTCA AT - #ATGGTAGT     480    # 522              GACA CTGTAGGTTC CCGGGTGCCT GC    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  597 nuc - #leotides              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  cDNA    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #16:    - GGAATCTTTT CTCACAACGA TGTTGAGGAA GTTAGAGATG TGATTTTGCC TC - #GAAATCCA      60    - GCCCGTGCAG TAGTAGGTGC CGCTGTCTGT TTCTGTGGCA TTGTTAATGG TG - #ATGTTGTA     120    - GTTGTCGTAC CAGAACTGGA GGAACTTGCC ATTCCTGTAG TAGGTCACTT TT - #TTGACATT     180    - CAAATTCCTC CAACTGTGGC ACCTGATGAG GAAGGACTCA CCCTCCAGCA CC - #ACCTCAGT     240    - AGAGGCCTGA AGGAGCAGCC ACTCACGGAA GACTTTTAAG GACACAGGTT TA - #CTCAGCAT     300    - GGATCCTTTG TTCTGACACG TGTATTCCCC ACTATCGCGG ATTTGGGCTT TC - #ACGATGTC     360    - CAAAGTTAAA GTTGTCACTC CCAACTTGGT CTTGTTGTGG AACCACACAG TA - #GAGTTGAG     420    - TTCAAGAGAA TTGTTCTCTT TACATGTAAG AGTCACACTG TCTTCTTTCA AT - #ATGGTAGT     480    - CCACGGTGGA TTCAAGGACA CTGTAGGTTC CCGGGTGCCT GCTGACATGC CA - #TCTGGATA     540    - GAGGAGGAGC AGCAGTGCTG TCCACAGCAG AGCAGGGCCT CCCAGGAAAA CC - #GGCAT     597    __________________________________________________________________________

What is claimed is:
 1. An isolated feline Fc.sub.ε Rα protein, whereinsaid isolated protein is isolated from a cell with which it naturallyoccurs, and wherein said protein is selected from the group consistingof:(a) a protein that (i) is encoded by a nucleic acid molecule that isat least about 80% identical to a member selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:11 andSEQ ID NO:14, wherein said percent identity is calculated according toDNAsis™ algorithms using default prarameters, and ii) has a functionselected from the group consisting of the ability to bind to an antibodythat binds to a naturally-occurring feline Fc.sub.ε Rα protein and theability to bind to IgE; (b) a protein comprising at least one epitope ofthe soluble domain of the protein of (a), wherein said epitope is ableto bind to an antibody that binds to a naturally-occurring felineprotein; and (c) a protein comprising at least one IgE binding domain ofthe protein of (a), wherein said domain is able to bind to IgE.
 2. Theprotein of claim 1, wherein said protein is selected from the groupconsisting of: a protein encoded by a nucleic acid molecule having anucleic acid sequence selected from the group consisting of: SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:14; and aprotein encoded by a nucleic acid molecule comprising a felinenaturally-occurring allelic variant of a nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:14. 3.The protein of claim 1, wherein said protein is selected from the groupconsisting of: a protein comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12 and SEQID NO:13; and a protein encoded by a feline naturally-occurring allelicvariant of a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:14.
 4. The protein of claim 1,wherein said feline FcεRα protein comprises a protein selected from thegroup consisting of PfelFc.sub.ε Rα₂₃₈, PfelFc.sub.ε Rα₂₆₃, PfelFc.sub.εRα₁₉₉ and PfelFc.sub.ε Rα₁₇₄.
 5. A therapeutic composition that, whenadministered to an animal, reduces Fc epsilon receptor-mediatedbiological responses, said therapeutic composition comprising anisolated soluble portion of a feline Fc.sub.ε Rα protein that is solubleand binds to IgE and an excipient, wherein said protein is selected fromthe group consisting of:(a) a protein that (i) is encoded by a nucleicacid molecule that is at least about 80% identical to a member selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:11 and SEQ ID NO:14, wherein said percent identity is calculatedaccording to DNAsis™ algorithms using default parameters, and (ii) has afunction selected from the group consisting of the ability to bind to anantibody that binds to a feline Fc.sub.ε Rα protein and the ability tobind to IgE; (b) a protein comprising at least one epitope of thesoluble domain of the protein of (a), wherein said epitope is able tobind to an antibody that binds to a naturally-occurring feline protein;and (c) a protein comprising at least one IgE binding domain of theprotein of (a) wherein said domain is able to bind to IgE.
 6. Thecomposition of claim 5, wherein said soluble portion of a felineFc.sub.ε Rα protein comprises a peptide that is a portion of a felineFc.sub.ε Rα protein that binds to IgE.
 7. The composition of claim 5,wherein said composition further comprises a component selected from thegroup consisting of an adjuvant and a carrier.
 8. A method to reduce Fcepsilon receptor-mediated biological responses in an animal comprisingadministering to an animal an effective amount of a therapeuticcomposition comprising an isolated soluble portion of a feline Fc.sub.εRα protein that is soluble and binds to IgE and an excipient, whereinsaid protein is selected from the group consisting of:(a) a protein that(i) is encoded by a nucleic acid molecule that is at least about 80%identical to a member selected from the group consisting of SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:14, wherein saidpercent identity is calculated according to DNAsis algorithms usingdefault parameters, and (ii) has a function selected from the groupconsisting of the ability to bind to an antibody that binds to a felineFc.sub.ε Rα protein and the ability to bind to IgE; (b) a proteincomprising at least one epitope of the soluble domain of the protein of(a), wherein said epitope is able to bind to an antibody that binds to anaturally-occurring feline protein; and (c) a protein comprising atleast one IgE binding domain of the protein of (a), wherein said domainis able to bind to IgE.
 9. The method of claim 8, wherein said solubleportion of a feline Fc.sub.ε Rα protein comprises a peptide of a felineFc.sub.ε Rα protein that binds to IgE.
 10. The method of claim 8,wherein said composition further comprises a component selected from thegroup consisting of an adjuvant and a carrier.
 11. An isolated felineFc.sub.ε Rα protein, wherein said isolated protein is isolated from acell with which it naturally occurs, and wherein said protein isselected from the group consisting of:(a) a protein (i) having an aminoacid sequence that is at least about 70% identical to a member selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:12 andSEQ ID NO:13, wherein said percent identity is calculated according toDNAsis algorithms using default parameters, and (ii) having a functionselected from the group consisting of the ability to bind to an antibodythat binds to a naturally-occurring feline Fc.sub.ε Rα protein and theability to bind to IgE; (b) a protein comprising at least one epitope ofthe soluble domain of the protein of (a), wherein said epitope is ableto bind to an antibody that binds to a naturally-occurring felineprotein; and (c) a protein comprising at least one IgE binding domain ofthe protein of (a), wherein said domain is able to bind to IgE.
 12. Atherapeutic composition that, when administered to an animal reduces Fcepsilon receptor-mediated biological responses, said therapeuticcomposition comprising an isolated soluble portion of a feline Fc.sub.εRα protein that is soluble and binds to IgE and an excipient, whereinsaid protein is selected from the group consisting of:(a) a protein (i)having an amino acid sequence that is at least about 70% identical to amember selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7,SEQ ID NO:12 and SEQ ID NO:13, wherein said percent identity iscalculated according to DNAsis algorithms using default parameters and(ii) having a function selected from the group consisting of the abilityto bind to an antibody that binds to a naturally-occurring felineFc.sub.ε Rα protein and the ability to bind to IgE; (b) a proteincomprising at least one epitope of the soluble domain of the protein of(a), wherein said epitope is able to bind to an antibody that binds to anaturally-occurring feline protein; and (c) a protein comprising atleast one IgE binding domain of the protein of (a), wherein said domainis able to bind to IgE.
 13. A method to reduce Fc epsilonreceptor-mediated biological responses in an animal comprisingadministering to an animal an effective amount of a therapeuticcomposition comprising an isolated soluble portion of a feline Fc.sub.εRα protein that is soluble and binds to IgE and an excipient, whereinsaid protein is selected from the group consisting of:(a) a protein (i)having an amino acid sequence that is at least about 70% identical to amember selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7,SEQ ID NO:12 and SEQ ID NO:13, wherein said percent identity iscalculated according to DNAsis algorithms using default parameters, and(ii) having a function selected from the group consisting of the abilityto bind to an antibody that binds to a naturally-occurring felineFc.sub.ε Rα protein and the ability to bind to IgE; (b) a proteincomprising at least one epitope of the soluble domain of the protein of(a), wherein said epitope is able to bind to an antibody that binds to anaturally-occurring feline protein; and (c) a protein comprising atleast one IgE binding domain of the protein of (a), wherein said domainis able to bind to IgE.